Small Molecule Ice Recrystallization Inhibitors And Methods Of Use Thereof

ABSTRACT

Ice recrystallization inhibitor (IRI) compounds and methods for cryopreserving umbilical cord blood are provided. The compounds are unsubstituted, mono-substituted, or di-substituted aryl-adlonamides. The methods include fractionating whole umbilical cord blood to generate a fraction comprising hematopoietic stem cells, mixing the hematopoietic stem cells with at least one IRI compound to form an IRI suspension, and freezing the IRI suspension.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. application Ser. No.14/403,391 filed on Nov. 24, 2014, which is a National Phase Applicationunder 35 U.S.C. §371 of International Application No. PCT/IB2013/001043filed on May 24, 2013, which claims the benefit of priority of U.S.Provisional Application No. 61/651,405 filed on May 24, 2012. The entiredisclosures of the above applications are incorporated herein byreference.

FIELD

The present invention relates to compounds with ice recrystallizationinhibition activity and methods of cryopreserving biological materialusing such compounds.

BACKGROUND

Cellular injury resulting from freezing and thawing has been studied forover sixty years. Under slow cooling conditions ice will form outside ofthe cell, concentrating solutes and creating an osmotic flux. Cells withless permeable membranes will incur increasing osmotic pressureresulting in intracellular ice formation and cell rupture if dehydrationof the cell cannot occur. However, rapid dehydration may also be lethalto the cell and cryoprotectants are employed to mitigate cellulardamage. Cryoprotectants are cytotoxic, and the mechanism ofintracellular ice formation and subsequent cell death are not wellunderstood. While the formation of intracellular ice correlates withcell death, it does not directly kill cells. Rather, the process of icerecrystallization (a form of ice crystal re-modeling that occurs duringwarming), is believed to be a significant factor contributing to celldeath. The importance of ice recrystallization as a mechanism ofcellular damage is supported by the fact that (1) freeze-tolerantorganisms inhabiting sub-zero environments produce large quantities ofrecrystallization inhibitors in vivo to ensure survival and (2) icerecrystallization damages cell membranes during cryopreservation.

Stem cell and regenerative therapy using cryopreserved umbilical cordblood is hampered by decreased cell function and viability afterthawing. Consequently, improved cryopreservation protocols that increasethe yield of viable and functional cells are urgently required. Dimethylsulfoxide (DMSO) is currently regarded as the “gold standard” forcryopreservation of stem cells and umbilical cord blood. While allcryoprotectants are potentially cytotoxic in vitro, DMSO has exhibitedsignificant cytotoxic effects in the clinical setting. Variousbiopolymers have been explored as an alternative to DMSO, but fail toprovide the high cell viabilities observed with DMSO or glycerol.Similarly, various sugars (mono- and disaccharides) have also beeninvestigated as cryoprotectants. However, the structure of thecarbohydrate, the freezing protocol, cell type and reported cellviabilities vary dramatically between studies making it difficult toascertain the true ability of these compounds to protect cells againstcryo-injury. To date, a viable alternative to DMSO, has not yet beenidentified.

Improved cryopreservation compositions and methods have the potential torevolutionize solid organ transplantation by allowing organs such aslivers and kidneys to be successfully preserved and transported morewidely.

The mechanism of recrystallization (both with inorganic materials andice) has been studied and several models have emerged.

Biological antifreezes (BAs) are a very interesting class of moleculescomprised of antifreeze proteins (AFPs) and antifreeze glycoproteins(AFGPs) which protect organisms inhabiting sub-zero environments fromcryo-injury and death. However, attempts to utilize BAs ascryoprotectants have been met with limited success. This is largelybecause BAs exhibit thermal hysteresis (TH) activity, meaning they havethe ability to selectively depress the freezing point of a solutionbelow that of the melting point. The TH activity associated with thesecompounds is a result of irreversible binding of BAs to the surface ofice, exacerbating cellular damage at cryopreservation temperatures. Thisis unfortunate as BAs are also potent inhibitors of icerecrystallization.

During the last decade, the rational design of novel compounds based onBAs has featured prominently in the literature. However, only a few ofthese molecules have the ability to inhibit ice recrystallization,limiting compounds with potent ice recrystallization inhibition activityto BAs (native AFPs and AFGPs), synthetic analogues of AFGPs andpolyvinyl alcohol (PVA). It has been demonstrated that C-linked AFGPanalogues 1 and 2, shown below

possess “custom-tailored” antifreeze activity. They are potentinhibitors of ice recrystallization but do not exhibit TH activity. Thecryoprotective ability of C-linked AFGP analogues 1 and 2 has also beenassessed, and analog 1 was found to be as effective as a 2.5% solutionof DMSO for the cryopreservation of human embryonic liver cells. It hasalso been demonstrated that simple mono- and disaccharides are moderateinhibitors of ice recrystallization and that inhibition of icerecrystallization during cryopreservation with human embryonic livercell lines and human umbilical cord blood leads to increased cellviability after thawing. While these simple carbohydrates classify assmall molecules, they are not potent inhibitors of icerecrystallization. Consequently, small molecules exhibiting potent IRIactivity are very attractive, but efforts to design such molecules havebeen impeded because the structural attributes necessary to inhibit icerecrystallization remain unknown.

SUMMARY

To date, there have been no reports of small molecule icerecrystallization inhibitor (IRI) compounds. Without being bound bytheory, it is believed that the moderate ice recrystallizationinhibition activity observed with simple carbohydrates is based upontheir ability to alter the structure of bulk water which is ultimatelyrelated to the degree of carbohydrate hydration. Thus, in an effort toidentify potent small molecule inhibitors of ice recrystallization, theability of various small molecules which alter the structure of bulkwater and/or sequester bulk water to inhibit ice recrystallization wereinvestigated. Such compounds include surfactants, organogelators andhydrogelators. The ability of representative low molecular weightcarbohydrate-based compounds to inhibit ice recrystallization wasassessed and it was demonstrated that these compounds are potent IRIcompounds.

In one embodiment, a composition for cryopreserving umbilical cord bloodis provided. The composition includes at least one ice recrystallizationinhibitor compound, wherein the ice recrystallization inhibitor compoundis a mono- or di-substituted aryl aldonamide.

In another embodiment, a method for cryopreserving umbilical cord bloodis provided. The method includes fractionating whole umbilical cordblood to generate a fraction having hematopoietic stem cells, mixing thehematopoietic stem cells in a solution including at least one icerecrystallization inhibitor compound to form an ice recrystallizationinhibitor suspension, and freezing the ice recrystallization inhibitorsuspension. The ice recrystallization inhibitor is an unsubstitutedaryl-aldonamide, a mono-substituted aryl-aldonamide, or a di-substitutedaryl-aldonamide.

In yet another embodiment, another method of cryopreservinghematopoietic stem cells is provided. The method includes adding an icerecrystallization inhibitor compound to a suspension of hematopoieticstem cells to form an ice recrystallization inhibitor suspension, andfreezing the ice recrystallization inhibitor suspension. The icerecrystallization inhibitor compound corresponds to Formula B′:

wherein R and R₂ are independently a C₁-C₄-alkoxy; or halo selected fromthe group consisting of Br, Cl, and F, and wherein neither R nor R₂ is aH.

Other embodiments, including particular aspects of the embodimentssummarized above, will be evident from the detailed description thatfollows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of ice recrystallization inhibitionactivity of compound 3 and compound 4, and anionic surfactant sodiumdodecyl sulphate (SDS). All compounds are represented as a % MGS (meangrain size) of ice crystals relative to the phosphate buffered saline(PBS) positive control.

FIG. 2 is the structures of D-sorbitol, D-dulcitol, low molecular weighthydrogelator derivatives N-octyl-D-gluconamide (compound 5) andN-octyl-D-galactonamide (compound 6), N-methylated derivatives(compounds 7 and 8), and ether-linked derivatives (compounds 9 and 10).

FIG. 3 is a graphical representation of ice recrystallization inhibitionactivity of D-sorbitol, D-dulcitol, and compounds 5-9. All compounds arerepresented as a % MGS (mean grain size) of ice crystals relative to thePBS positive control.

FIG. 4( a) shows the ice crystal habits of compound 3 assayed at aconcentration of 1 mg/mL.

FIG. 4( b) shows the ice crystal habits of compound 4 assayed at aconcentration of 1 mg/mL

FIG. 4( c) shows the ice crystal habits of compound 5 assayed at aconcentration of 0.01 mg/mL

FIG. 4( d) shows the ice crystal habits of compound 6 assayed at aconcentration of 0.01 mg/mL.

FIG. 4( e) shows the ice crystal habits of AFGP-8 assayed at aconcentration of 0.01 mg/mL in water.

FIG. 5( a) is a graphical representation of the saturation recoverycurves of frozen D₂O with the AFP controls, which were three positivecontrols for ice binding: Type I AFP (R₁=0.13±0.01 s⁻¹), Type III AFP(R₁=0.22±0.01 s⁻¹), WT LpAFP (R₁=0.039±0.002 s⁻¹); and one negativecontrol for ice binding: T67Y LpAFP (R₁=0.018±0.001 s⁻¹). The ²Hrelaxation rate for frozen D₂O was R₁=0.0154±0.0002 s⁻¹.

FIG. 5( b) is a graphical representation of the saturation recoverycurves of frozen D₂O with the AFP controls and compound 4(R₁=0.0209±0.0002 s⁻¹). The concentration at which compound 4 wasmeasured was 44 mM. The concentration compound 4 was corrected to atotal overall proton concentration of 1234 mM, unless stated otherwise.

FIG. 5( c) is a graphical representation of the saturation recoverycurves of frozen D₂O with the AFP controls and frozen hydrogel ofcompound 5 (R₁=0.0310±0.0005 s⁻¹). The concentration at which compound 5was measured was 42 mM. The concentration of compound 5 was corrected toa total overall proton concentration of 1234 mM, unless statedotherwise.

FIG. 5( d) is a graphical representation of the saturation recoverycurves of frozen D₂O with the AFP controls and frozen hydrogel ofcompound 6 (R₁=0.030±0.001 s⁻¹). The concentration at which compound 6was measured was 16 mM, which corresponds to a total overall protonconcentration of 479 mM.

FIG. 6 is a graphical representation of ice recrystallization inhibitionactivity of n-alkyl-gluconamides in 0.5 gm/mL NaCl.

FIG. 7 is a graphical representation of ice recrystallization inhibitionactivity of n-alkyl-erythronamides in 0.5 gm/mL NaCl.

FIG. 8 is a graphical representation of ice recrystallization inhibitionactivity of para-substituted aryl-glycosides derivatives in PBS.

FIG. 9 is a graphical representation of ice recrystallization inhibitionactivity of glucose derivatives with substituents at the anomericposition in PBS.

FIG. 10 is a graphical representation of ice recrystallizationinhibition activity of aryl mono- and disaccharides in PBS.

FIG. 11 is a graphical representation of ice recrystallizationinhibition activity of C₆—OH modified glycoside derivatives in PBS.

FIG. 12 is a graphical representation of the HepG2 cell viability ofPMP-glucoside.

FIG. 13 is a graphical representation of the HepG2 cell viability ofn-octyl-gluconamide.

FIG. 14( a) is a graphical representation of the saturation recoverycurves of frozen D₂O with the AFP controls and D-galactose(R₁=0.0154±0.0003 s⁻¹). The concentration at which D-galactose wasmeasured was 103 mM. The concentration of D-galactose was corrected to atotal overall proton concentration of 1234 mM, unless stated otherwise.

FIG. 14( b) is a graphical representation of the saturation recoverycurves of frozen D₂O with the AFP controls and D-glucose(R₁=0.0132±0.0001 s⁻¹). The concentration at which D-glucose wasmeasured was 103 mM. The concentration of D-D-glucose was corrected to atotal overall proton concentration of 1234 mM, unless stated otherwise.

FIG. 14( c) is a graphical representation of the saturation recoverycurves of frozen D₂O with the AFP controls and compound 3(R₁=0.0190±0.0002 s⁻¹). The concentration at which compound 3 wasmeasured was 44 mM. The concentration of compound 3 was corrected to atotal overall proton concentration of 1234 mM, unless stated otherwise.

FIG. 14( d) is a graphical representation of the saturation recoverycurves of frozen D₂O and WT LpAFP (R₁=0.0133±0.0005 s⁻¹). Theconcentration at which WT LpAFP was measured was 0.5 mM, whichcorresponds to a total overall proton concentration of 433 mM.

FIG. 15( a) is a graphical representation showing % intact (100-%hemolysis) v. temperature (° C.) for cryoprotectant solutions of PMP-Glcand glycerol as well as glycerol alone used to preserve red blood cells(RBCs) during slow cooling conditions.

FIG. 15( b) is a graphical representation showing % intact (100-%hemolysis) for cryoprotectant solutions of PMP-Glc and glycerol as wellas glycerol alone used to preserve RBCs during slow cooling conditions.

FIG. 16( a) is a graphical representation showing % intact (100-%hemolysis) v. temperature (° C.) for cryoprotectant solutions of PMP-Glcand glycerol as well as glycerol alone used to preserve RBCs duringrapid cooling conditions.

FIG. 16( b) is a graphical representation showing % intact (100-%hemolysis) for cryoprotectant solutions of PMP-Glc and glycerol as wellas glycerol alone used to preserve RBCs during slow rapid conditions.

FIG. 17( a) is a graphical representation showing % intact (100-%hemolysis) v. temperature (° C.) for cryoprotectant solutions of PMP-Glcand glycerol as well as glycerol alone used to preserve RBCs during slowcooling conditions and incubation at room temperature or 0° C.

FIG. 17( b) is a graphical representation showing % intact (100-%hemolysis) for cryoprotectant solutions of PMP-Glc and glycerol as wellas glycerol alone used to preserve RBCs during slow cooling conditionsand incubation at room temperature or 0° C.

FIG. 18( a) is a graphical representation showing % intact (100-%hemolysis) v. temperature (° C.) for cryoprotectant solutions of PMP-Glcand glycerol as well as glycerol alone used to preserve RBCs duringrapid cooling conditions and incubation at room temperature or 0° C.

FIG. 18( b) is a graphical representation showing % intact (100-%hemolysis) for cryoprotectant solutions of PMP-Glc and glycerol as wellas glycerol alone used to preserve RBCs during rapid cooling conditionsand incubation at room temperature or 0° C.

FIG. 19( a) is a graphical representation showing % intact (100-%hemolysis) v. temperature (° C.) for cryoprotectant solutions of sucroseand glycerol as well as glycerol alone used to preserve RBCs during slowand rapid cooling conditions.

FIG. 19( b) is a graphical representation showing % intact (100-%hemolysis) for cryoprotectant solutions of sucrose and glycerol as wellas glycerol alone used to preserve RBCs during slow and rapid coolingconditions.

FIG. 20( a) is a graphical representation showing % intact (100-%hemolysis) v. temperature (° C.) for cryoprotectant solutions offructose and glycerol as well as glycerol alone used to preserve RBCsduring slow and rapid cooling conditions.

FIG. 20( b) is a graphical representation showing % intact (100-%hemolysis) for cryoprotectant solutions of fructose and glycerol as wellas glycerol alone used to preserve RBCs during slow and rapid coolingconditions.

FIG. 21( a) is a graphical representation showing % intact (100-%hemolysis) v. temperature (° C.) for cryoprotectant solutions of OGG-Galand glycerol as well as glycerol alone used to preserve RBCs during slowand rapid cooling conditions.

FIG. 21( b) is a graphical representation showing % intact (100-%hemolysis) for cryoprotectant solutions of OGG-Gal and glycerol as wellas glycerol alone used to preserve RBCs during slow and rapid coolingconditions.

FIG. 22 is a graphical representation showing % intact (100-% hemolysis)for for cryoprotectant solutions PMP-Glc (compound 17), pFPh-Glc(Compound 16), pBrPh-Glc (compound 14) and glycerol as well as glycerolalone used to preserve RBCs post-thaw during incubation with thecryoprotectant solution at room temperature and slow cooling to −40° C.and −50° C.

FIG. 23 is a graphical representation showing % intact (100-% hemolysis)for cryoprotectant solutions PMP-Glc (compound 17), pFPh (Compound 16),pBrPh-Glu (compound 14) and glycerol as well as glycerol alone used topreserve RBCs post-thaw during incubation with the cryoprotectantsolution at room temperature and slow cooling to −40° C. and −50° C.,and storage at −80° C. for 2 hours.

FIG. 24 is a graphical representation of ice recrystallizationinhibition activity of aryl-glycosides in PBS, including PMP-Glc,pFPh-Glc and pBrPh-Glc. All compounds are represented as a % MGS (meangrain size) of ice crystals relative to the phosphate buffered saline(PBS) positive control

FIG. 25 is a graphical representation of the HepG2 cell viability with acryoprotectant solution of PMP-glucose, pFPh-Glc and pBrPh-Glc atdifferent concentrations.

FIG. 26( a) is a graphical representation of Tfl-α (or Tfl-a) cellviability with 0%, 2% or 10% DMSO.

FIG. 26( b) is a graphical representation of Tfl-α cell viability forcryoprotectant solutions, each containing compounds 1a, 2a and 3a at 55mM and 0%, 2% or 10% DMSO.

FIG. 27( a) is a reaction scheme for the synthesis ofN-(4-chlorophenyl)-D-gluconamide.

FIG. 27( b) is a reaction scheme for the synthesis ofN-(2,6-difluorobenzyl)-D-gluconamide.

FIG. 28 is a graph showing ice recrystallization inhibition activity ofvarious compounds.

FIG. 29 is a graph showing CD34+ post-thaw viability forcryopreservation of stem cells with various concentrations ofN-(4-methoxyphenyl)-D-gluconamide.

FIG. 30 is a graph showing CD34+ post-thaw viability forcryopreservation of stem cells with various concentrations ofN-(2-fluorophenyl)-D-gluconamide.

FIG. 31 is a graph showing CD34+ post-thaw viability forcryopreservation of stem cells with various concentrations ofN-(2,6-difluorobenzyl)-D-gluconamide.

FIG. 32 is a graph showing CD34+ post-thaw viability forcryopreservation of stem cells with various concentrations ofN-(4-chlorophenyl)-D-gluconamide.

FIG. 33 is a graph showing the total number of colonies formed aftercryopreservation with N-(4-methoxyphenyl)-D-gluconamide at variousconcentrations post-thaw.

FIG. 34 is a graph showing the total number of colonies formed aftercryopreservation with N-(2-fluorophenyl)-D-gluconamide at variousconcentrations post-thaw.

FIG. 35 is a graph showing the total number of colonies formed aftercryopreservation with N-(2,6-difluorobenzyl)-D-gluconamide at variousconcentrations post-thaw.

FIG. 36 is a graph showing the total number of colonies formed aftercryopreservation with N-(4-chlorophenyl)-D-gluconamide at variousconcentrations post-thaw.

FIG. 37 is a graph showing the cytotoxicity ofN-(4-methoxyphenyl)-D-gluconamide.

FIG. 38 is a graph showing the cytotoxicity ofN-(2-fluorophenyl)-D-gluconamide.

FIG. 39 is a graph showing the cytotoxicity ofN-(2,6-difluorobenzyl)-D-gluconamide.

FIG. 40 is a graph showing the cytotoxicity ofN-(4-chlorophenyl)-D-gluconamide.

FIG. 41 is a graph showing the combined results of a colony formingunits assay, where a horizontal line shows the level of a control.

DETAILED DESCRIPTION

IRI compounds, compositions and kits for cryopreservation, and methodsfor cryopreserving a biological material are provided herein.

I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention belongs. In case of conflict, the presentapplication including the definitions will control. All publications,patents and other references mentioned herein are incorporated byreference in their entireties for all purposes as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

Although methods and materials similar or equivalent to those describedherein can be used in practice or testing of the present invention,suitable methods and materials are described below. The materials,methods and examples are illustrative only and are not intended to belimiting. Other features and advantages of the invention will beapparent from the detailed description and from the claims.

To facilitate an understanding of the present invention, a number ofterms and phrases are defined below.

As used in the present disclosure and claims, the singular forms “a,”“an,” and “the” include plural forms unless the context clearly dictatesotherwise.

Wherever embodiments are described herein with the language“comprising,” otherwise analogous embodiments described in terms of“consisting of” and/or “consisting essentially of” are also provided.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include “A and B”, “A or B”, “A”, and “B”.

The term “aldonamide” refers to an amide of an aldonic acid, and theterm “aldonic acid” refers to a sugar substance in which the aldehydegroup (generally found at the C1 position on the sugar) has beenreplaced by a carboxylic acid. Aldonamides may be based on compoundscomprising one saccharide unit, two saccharide units or they may bebased on compounds compromising more than two saccharide units as longas the polysaccharide has a terminal sugar unit with an aldehyde groupavailable for oxidation to a carboxylic acid group. Examples of analdonamide based on one saccharide unit include, but are not limited to,ribonamide, gluconamide, and glucoheptonamide. Examples of an aldonamidebased on two saccharide units include, but are not limited to,lactobionamide and maltobionamide. The aldonamide may be substituted orunsubstituted with groups, such as, but not limited to an alkyl groupand an aryl group.

The term “alkoxy” refers to straight-chain or branched alkyl grouphaving 1 to about 8 carbons bonded to an oxygen, such as, but notlimited to, methoxy, ethoxy, propoxy, butoxy, pentoxy, hexoxy, heptoxyand octoxy. The term “C₁-C₄-alkoxy” refers to an alkoxy having 1 to 4carbon atoms such as, but not limited to, methoxy, ethoxy, n-propoxy,1-methylethoxy, n-butoxy, 1-methylpropoxy, 2-methylpropoxy and1,1-dimethylethoxy. “Alkoxy” is intended to embrace all structuralisomeric forms of an alkoxy group. For example, as used herein, propoxyencompasses both n-propoxy and isopropoxy, etc.

The term “alkyl” refers to a saturated hydrocarbon chain of 1 to about 8carbon atoms in length, such as, but not limited to, methyl, ethyl,propyl and butyl. The alkyl group may be straight-chain orbranched-chain. The term ““C₁-C₄-alkyl” as used refers to a saturatedstraight-chain or branched hydrocarbon having 1 to 4 carbon atoms.“Alkyl” is intended to embrace all structural isomeric forms of an alkylgroup. For example, as used herein, propyl encompasses both n-propyl andisopropyl; butyl encompasses n-butyl, sec-butyl, isobutyl andtert-butyl.

The term “aryl” refers to a monocyclic or polycyclic aromatic group,such as, but not limited to, phenyl, napthyl, thienyl and indolyl.

The term “biological material” refers to any substance which can or hasto be removed from a human or non-human, such as an animal, body that issuitable for cryopreservation, such as, but not limited to, organs,tissues, cells, sperm, eggs and embryos. Examples of cells include, butare not limited to, a cell line, a stem cell, a progenitor cell, a livercell and a red blood cell.

The term “carbohydrate” refers to a compound consisting of carbon,hydrogen, and oxygen. Examples include, but are not limited to,monosaccharides, disaccharides, oligosaccharides and polysaccharides aswell as substances derived from monosaccharides by reduction of thecarbonyl group (alditols), by oxidation of one or more terminal groupsto carboxylic acids, or by replacement of one or more hydroxy group(s)by a hydrogen atom, an amino group, a thiol group or similarheteroatomic groups. “Carbohydrate” is intended to encompass unmodifiedcarbohydrates, carbohydrate derivatives, substituted carbohydrates, andmodified carbohydrates. As used herein, the phrases “carbohydratederivatives”, “substituted carbohydrate”, and “modified carbohydrates”are synonymous. Modified carbohydrate means any carbohydrate wherein atleast one atom has been added, removed, substituted, or combinationsthereof. Thus, carbohydrate derivatives or substituted carbohydratesinclude substituted and unsubstituted monosaccharides, disaccharides,oligosaccharides, and polysaccharides.

The term “cell medium” refers to a liquid or gel designed to support thegrowth of microorganisms or cells, such as, but not limited to, Eagle'sMinimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium(DMEM), Roswell Park Memorial Institute medium (RPMI), Fetal BovineSerum (FBS), Fetal Calf Serum (FCS), Ham's F-10, Ham's F-12, Hank'sbuffered salt solution (HBSS), HBSS and dextrose, and Medium 199.

The term “cryopreservation agent” refers to a compound which assists inthe cryopreservation of a biological material. Examples of suitablecryopreservation agents include, but are not limited to, DMSO, glycerol,and other biopolymers used in cryopreservation. Examples of suitablebiopolymers include, but are not limited to, polyvinyl alcohol.“Cryopreservation agent” as used herein does not include water, RPMI,DMEM, MEM and HBSS and dextrose.

The term “erythronamide” refers to an amide of erythronic acid, and theterm “erythronic acid” refers the acid of the carbohydrate, erythrose(C₄H₈O₄). An erythronamide can be substituted with groups such as, butnot limited to, an alkyl and a hydroxyalkyl.

The term “halogen” including derivative terms, such as “halo” refers tofluorine, chlorine, bromine and iodine.

The term “haloalkyl” refers to a straight-chain or branched alkyl groupsubstituted with from 1 to the maximum possible number of halogen atoms.The term “C₁-C₄-haloalkyl” refers to a straight-chain or branched alkylgroup having 1 to 4 carbon atoms, where some or all of the hydrogenatoms in the alkyl groups may be replaced by fluorine, chlorine, bromineand/or iodine. Examples of a C₁-C₄-haloalkyl include, but are notlimited to, chloromethyl, dichloromethyl, trichloromethyl, fluoromethyl,difluoromethyl, trifluoromethyl, chlorofluoromethyl,dichlorofluoromethyl and chlorodifluoromethyl.

The term “glycoside” refers to a molecule in which a sugar group isbonded through its anomeric carbon to another group via a glycosidicbond. A “glycosidic bond” may be an O— (an O-glycoside), N— (aglycosylamine), S— (a thioglycoside), or C— (a C-glycoside) bond. Thesugar group in a glycoside may be a monosaccharide or anoligosaccharide. The sugar group in a glycoside may be bonded to groupssuch as, but not limited to, an alkyl group and an aryl group

The term “monosaccharide” refers to a simple sugar which upon hydrolysisdoes not break down into a smaller simple sugar. “Monosaccharide” isintended to encompass an aldose and a ketose. An “aldose” refers to asimple sugar that contains only one aldehyde group per molecule. A“ketose” refers to a simple sugar that contains one ketone group permolecule. Examples of monosaccharides include, but are not limited to,glucose, fructose, galactose, xylose and ribose.

The terms “oligosaccharide” and “polysaccharide” refer to compoundsconsisting of monosaccharides linked glycosidically. In generalpolysaccharides comprise at least 10 monosaccharide residues, whereasoligosaccharides in general comprise in the range of 2 to 20monosaccharides. Oligosaccharides and polysaccharides may be linear orbranched.

The term “polyvinyl alcohol (PVA)” refers to water soluble polyhydroxycompounds which can be generally characterized, for instance, by thepresence of (—CH₂—CHOH—) units in a polymer chain. “Polyvinyl alcohol”includes all suitable grades, degrees of saponification and degrees ofpolymerization. Examples of polyvinyl alcohols include, but are notlimited to, grades of Mowiol®.

The term “pyranose” refers to carbohydrates having a chemical structurethat includes a six-membered ring consisting of five carbon atoms andone oxygen atom. For example the sugars including, but not limited to,allose, altrose, glucose, galactose and mannose can be in pyranose form.

The term “sugar” refers to monosaccharides, oligosaccharides,polysaccharides, as well as compounds comprising monosaccharide,oligosaccharide, or polysaccharide. The terms “carbohydrate” and “sugar”are herein used interchangeably.

II. IRI Compounds

In one embodiment, the IRI compound includes a hydrophilic carbohydrateresidue linked by an amide bond, ester, or ether group to a hydrophobicstructure, which may include an alkyl chain.

In another embodiment, the IRI compound can be derived from a pyranoseor open chain carbohydrate as a highly-hydrated group linked at C1 by anamide bond, acetyl or ether group to an alkyl chain or functionalizedalkyl chain.

In another embodiment, the IRI compound can include a carbohydrate-basednon-ionic surfactant. The carbohydrate-based non-ionic surfactants caninclude alkyl-glycosides, such as, but not limited ton-octyl-β-D-glycosides. Examples of suitable n-octyl-β-D-glycosidesinclude, but are not limited to, n-octyl-β-D-glucopyranoside (compound3) and n-octyl-β-D-galactopyranoside (compound 4) below.

In another embodiment, the IRI compound is a carbohydrate-basedhydrogelator. Carbohydrate-based hydrogelators can includen-alkyl-aldonamides such as, but not limited to n-octyl-D-aldonamides.An example of a suitable n-octyl-D-aldonamide is n-octyl-D-gluconamide(compound 5) below.

In another embodiment, the IRI compound is a n-alkyl-gluconamiderepresented by Formula I:

wherein n=0, 1, 2, 3, 5, 6, 7, or 8. Non-limiting examples ofn-alkyl-gluconamides include n-methyl-gluconamide (NMeGlc),n-ethyl-gluconamide (NEtGlc), n-propyl-gluconamide (NPrGlc),n-butyl-gluconamide (NBuGlc), n-pently-gluconamide (NPentGlc),n-hexyl-gluconamide (NHexGlc), n-heptyl-gluconamide (NHepGlc),n-octyl-gluconamide (NOGlc), n-nonyl-gluconamide (NNonGlc), and(N-Oct-OH-GLC). ice recrystallization inhibition activity for the listedn-alkyl-gluconamides is shown in FIG. 6.

In another embodiment, the IRI compound is a n-alkyl-erythronamide or an-hydroxyalkyl-erythronamide represented by Formula II:

wherein X is —CH₃ or —OH and n=3, 4, 5, 6, 7, 8. Non-limiting examplesof n-alkyl-erythronamides include n-butyl-erythronamide (NBuEryth),n-pentyl-erythronamide (NPentEryth), n-hexyl-erythronamide (NHexEryth),n-heptyl-erythronamide (NHepEryth), and n-octyl-erythronamide (NOEryth).Ice recrystallization inhibition activity for the listedn-alkyl-erythronamides is shown in FIG. 7. Non-limiting examples of an-hydroxyalkyl-erythronamides include, for example,n-butanol-erythronamide, n-pentenol-erythronamide,n-hexanol-erythronamide, n-heptanol-erythronamide, andn-octanol-erythronamide.

In another embodiment, the IRI compound is a para-, ortho- ormeta-substituted aryl-glycoside. Non-limiting examples ofpara-substituted aryl-glycosides are shown below.

Ice recrystallization inhibition activity for the para-substitutedaryl-glycoside derivatives is shown in FIG. 8. In another embodiment,the para-, ortho- or meta-substituted aryl-glycoside is a para-, ortho-or meta-substituted aryl-glucoside corresponding in structure to FormulaA below:

wherein R is hydrogen, halo, alkoxy, haloalkyl, alkyl or —NO₂.

In another embodiment, R is hydrogen; or halo selected from the groupconsisting of Br, Cl, and F; or C₁-C₄-alkoxy; or C₁-C₄-haloalkyl; orC₁-C₄-alkyl; or —NO₂

In another embodiment, R is hydrogen; or halo selected from the groupconsisting of Br, Cl, and F; or C₁-C₄-alkoxy.

In another embodiment, R is para-substituted.

In another embodiment, R is halo selected from the group consisting ofBr and F; or R is methoxy, wherein R is para-substituted.

Examples of the para-, ortho- or meta-substituted aryl-glucosidescorresponding in structure to Formula A are para-methoxy-phenylglucoside (PMP-Glc) (compound 17), para-fluoro-phenyl glucoside(pFPh-Glc) (compound 16) and para-bromo-phenyl glucoside (PBrPh-Glc)(compound 14).

Ice recrystallization inhibition activity for PMP-Glc, pFPh-Glc andpBrPh-Glc is shown in FIG. 24.

In another embodiment, the IRI compound is an aryl-aldonamide, such as,but not limited to a phenyl-aldonamide. The phenyl-aldonamide can be acompound corresponding in structure to Formula B:

wherein R is hydrogen; a C₁-C₄-alkoxy; or halo selected from the groupconsisting of Br, Cl, and F.

In another embodiment, R is hydrogen; or R is a para-, ortho- ormeta-substituted C₁-C₄-alkoxy; or R is an ortho-substituted haloselected from the group consisting of Br, Cl, and F.

In another embodiment, R is hydrogen; or R is a para-substitutedC₁-C₄-alkoxy; or R is an ortho-substituted halo selected from the groupconsisting of Br, Cl, and F.

In another embodiment, R is hydrogen; or R is a para-substitutedmethoxy; or R is an ortho-substituted F.

Examples of compounds corresponding in structure to Formula B are

However, some of the compounds shown above function as IRI compoundsbetter than others. In other words, some of the above compounds have ahigher IRI activity than others.

Compound 4a, N-(4-chlorophenyl)-D-gluconamide, for example, issynthesized by combining D-gluconic acid-d-lactone with 4-chloroanilinein acetic acid to form a mixture. The D-gluconic acid-d-lactone and4-chloroaniline are combined at a D-gluconicacid-d-lactone:4-chloroaniline ratio of from about 5:1 to about 1:5. Forexample, in various embodiments the D-gluconicacid-d-lactone:4-chloroaniline ratio is about 5:1, about 4:1, about 3:1,about 2:1, about 1.5:1, about 1:1, about 1:1.5, about 1:2, about 1:3,about 1:4, or about 1:5. The mixture is stirred under reflux for fromabout 10 minutes to about 24 hours. Then, crudeN-(4-chlorophenyl)-D-gluconamide is precipitated with hexanes, filtered,and recrystallized in ethanol to afford white crystals having aN-(4-chlorophenyl)-D-gluconamide yield of greater than or equal to 30%,or from about 30% to about 95%.

In another embodiment, the IRI compound is an aryl-aldonamide, such as,but not limited to an unsubstituted, mono-substituted, or di-substitutedbenzyl-aldonamide. The benzyl-aldonamide can be a compound correspondingin structure to Formula B′:

wherein R and R₂ are independently a hydrogen; C₁-C₄-alkoxy; or haloselected from the group consisting of Br, Cl, and F, and n is a wholenumber from 1 to 20 and represents the number of carbon atoms betweenthe benzyl group and the amide bond.

Examples of compounds corresponding in structure to Formula B′ are

However, compound (1b) functions as a IRI compound better than compound(2b). In other words, compound (1b) has a higher IRI activity thancompound (2b).

Compound 1b, N-(2,6-difluorobenzyl)-D-gluconamide, for example, issynthesized by combining D-gluconic acid-d-lactone with2,6-difluoroaniline in a solvent to generate a mixture. The D-gluconicacid-d-lactone and 2,6-difluooaniline are combined at a D-gluconicacid-d-lactone:2,6-difluooaniline ratio of from about 5:1 to about 1:5.For example, in various embodiments the D-gluconicacid-d-lactone:2,6-difluooaniline ratio is about 5:1, about 4:1, about3:1, about 2:1, about 1.5:1, about 1:1, about 1:1.5, about 1:2, about1:3, about 1:4, or about 1:5. The solvent is an alcohol, such asmethanol, ethanol, propanol, isopropanol, butanol, or isobutanol. Themixture is stirred under reflux for from about 12 hours to about 48hours. Then, the solvent is evaporated to generate a residue. Theresidue is recrystallized, for example, in ethanol, to form whitecrystals having a N-(2,6-difluorobenzyl)-D-gluconamide yield of greaterthan or equal to 50%, or from about 50% to about 95%.

In another embodiment, the IRI compound is a carbohydrate with apara-methoxy-phenyl (PMP) substituent at C1 (i.e. a PMP carbohydratederivative), wherein the carbohydrate is a pyranose or open chaincarbohydrate. In further embodiments, the anomeric oxygen may also bereplaced with a nitrogen atom.

In a particular embodiment, the IRI compound is a PMP carbohydratederivative, wherein the carbohydrate is a pyranose, preferably glucoseor galactose. An example of a suitable PMP carbohydrate derivative isPMP-glucoside (compound 17/PMP-Glc).

In a further particular embodiment, the IRI compound is a PMPcarbohydrate derivative, wherein the carbohydrate is a pyranose and thepyranose is linked to a second carbohydrate, preferably galactose, atthe C4 position, and preferably where the intersaccharidic linkage isbeta-, or equatorial.

In a further embodiment, the IRI compound is a PMP monosaccharidederivative represented by the Formula C below.

wherein PMP is

R₁-R₄ are each independently selected from H,

wherein n=0, 1, 2, 3, 4, 5, 6, 7 or 8, and each occurrence of R isindependently selected from the group consisting of a C₁-C₉-alkyl. Insome embodiments, at least one of R₁, R₂, R₃ and R₄ is OH.

In another embodiment, the IRI compound is a PMP carbohydratederivative, wherein the PMP substituent in the para-position can besubstituted with either bromine, chlorine, fluorine or iodine.

In another embodiment, the IRI compound is a glucose derivative with oneor more substituents at the anomeric position. Non-limiting examples ofglucose derivatives with a substituent at the anomeric position include:

Ice recrystallization inhibition activity for the glucose derivativeswith substituents at the anomeric position is shown in FIG. 9.

In another embodiment, the IRI compound is an aryl ring substituted withtwo or more mono- or disaccharides. Non-limiting examples of aryl ringmono- and disaccharides include:

Ice recrystallization inhibition activity for the aryl mono- anddisaccharides is shown in FIG. 10.

In another embodiment, the IRI compound is a C₆—OH modified glycosidederivative. Non-limiting examples of C₆—OH modified glycosidederivatives include glucose; xylose; glucuronic acid (compound 1d);6-deoxy-gluco-heptose(compound 2d); 1,6,-anhydro-glucose(compound 3d);galactose; fucose;(2R,3R,4S,5R,6R)-6-ethyltetrahydro-2H-pyran-2,3,4,5-tetraol (compound4d); (2R,3R,4S,5R,6R)-6-vinyltetrahydro-2H-pyran-2,3,4,5-tetraol(compound 5d); galacturonic acid (compound 6d); 6-deoxy-galacto-heptose(compound 7d); galacto-heptose (compound 8d) and 1,6-anhydro-galactose(compound 9d).

Ice recrystallization inhibition activity for the C₆—OH modifiedglycoside derivatives is shown in FIG. 11. In another embodiment, theIRI compound is a disaccharide derivative containing two pyranoses, twoopen chain carbohydrates, or a pyranose and an open chain carbohydrate,where the two pyranoses, the two open chain carbohydrates or thepyranose and the open chain carbohydrate are linked together with a PMPcompound or an alkyl chain.

III. Compositions for Cryopreserving Biological Material

Compositions for cryopreserving a biological material are providedherein comprising at least one IRI compound reported herein and at leastone cryopreservation agent reported herein.

In a particular embodiment, compositions for cryopreserving a biologicalmaterial are provided herein comprising at least one IRI compound,wherein the at least one IRI compound is selected from the groupconsisting of an alkyl-glycoside, a n-alkyl-aldonamide, an-alkyl-erythronamide, an aryl-glycoside, an aryl-aldonamide, and acombination thereof.

In a particular embodiment, compositions for cryopreserving a biologicalmaterial are provided herein, wherein the at least one IRI compound isan aryl-glycoside, an aryl-aldonamide or a combination thereof.

Preferably, the aryl-glycoside is a para-, ortho- or meta-substitutedaryl-glycoside, and preferably, the para-, ortho- or meta-substitutedaryl-glycoside is a para-, ortho- or meta-substituted aryl-glucosidecorresponding in structure to Formula A reported herein. Preferably, thearyl-glucoside corresponding in structure to Formula A ispara-substituted. Examples of suitable para-substituted aryl-glucosidesfor use in the compositions include, but are not limited to,para-methoxy-phenyl glucoside, para-fluoro-phenyl glucoside andpara-bromo-phenyl glucoside.

Preferably, the aryl-aldonamide is a phenyl-aldonamide corresponding instructure to Formula B reported herein. Examples of suitablephenyl-aldonamides for use in the composition include, but are notlimited to,

In a particular embodiment, the at least one IRI compound is present inthe composition in a concentration of less than about 400 mM, preferablyless than about 200 mM, preferably less than about 100 mM, preferablyless than about 10 mM, preferably less than about 1 mM, and preferablyless than about 0.5 mM. In another embodiment, the IRI compound can bepresent in the composition in a concentration of about 0.5 mM to (andincluding) about 400 mM, preferably about 55 mM to (and including) about220 mM.

In a particular embodiment, the para-, ortho- or meta-substitutedaryl-glycoside is present in a concentration between (and including)about 55 mM and about 110 mM.

In a particular embodiment, the aryl-aldonomide is present in thecomposition in a concentration between (and including) about 30 mM andabout 200 mM, and preferably about 55 mM.

In a particular embodiment, compositions for cryopreserving a biologicalmaterial are provided herein, wherein the at least one cryopreservationagent is selected from the group consisting of DMSO, glycerol, polyvinylalcohol, other biopolymers, or a combination thereof. The at least onecryopreservation agent is present in the composition in a concentrationof about 0.1% to (and including) about 30% v/v, preferably about 0.1% to(and including) about 20% v/v, preferably about 5% to (and including)about 30% v/v, and preferably about 5% to (and including) about 20% v/v.

In a particular embodiment, compositions for cryopreserving a biologicalmaterial are provided herein which further comprise a biologicalmaterial. Examples of biological material include, but are not limitedto, an organ, a tissue, and a cell. Examples of a cell include, but arenot limited to, a cell line, a stem cell, a progenitor cell, a livercell and a red blood cell, preferably the cell is a red blood cell, aliver cell or a progenitor cell.

In a particular embodiment, compositions for cryopreserving a biologicalmaterial are provided herein further comprising a cell medium. Examplesof suitable cell medium include Eagle's Minimum Essential Medium (MEM),Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park MemorialInstitute medium (RPMI), Fetal Bovine Serum (FBS), Fetal Calf Serum(FCS), Ham's F-10, Ham's F-12, Hank's buffered salt solution (HBSS),HBSS and dextrose, Medium 199, saline, dextran, plasma, and combinationsthereof.

In another embodiment, the compositions for cryopreservation can containboth biological material and cell medium as provided above.

IV. Kits for Cryopreserving Biological Material

Kits for cryopreserving a biological material are provided hereincomprising an IRI compound or composition for cryopreserving abiological material as reported herein. The IRI compound and a furthercryopreservation agent, if present, may be in the same composition or inseparation compositions. Additionally, they may be co-packaged forcommon presentation or packaged individually. Instructions can also beprovided in the kit for cryopreservation of various types of biologicalmaterial. The kits provided herein can further comprise a cell medium.Examples of suitable cell medium include Eagle's Minimum EssentialMedium (MEM), Dulbecco's Modified Eagle's Medium (DMEM), Roswell ParkMemorial Institute medium (RPMI), Fetal Bovine Serum (FBS), Fetal CalfSerum (FCS), Ham's F-10, Ham's F-12, Hank's buffered salt solution(HBSS), HBSS and dextrose, and Medium 199 and a combination thereof.

V. Methods for Cryopreserving Biological Material

Methods for cryopreserving a biological material are provided herein bysuspending the biological material in a solution of at least one IRIcompound reported herein to form a suspension and freezing thesuspension.

In a particular embodiment, methods for cryopreserving a biologicalmaterial are provided herein by suspending the biological material in asolution of at least one IRI compound, wherein the at least one IRIcompound is is selected from the group consisting of an alkyl-glycoside,a n-alkyl-aldonamide, a n-alkyl-erythronamide, an aryl-glycoside, anaryl-aldonamide, and a combination thereof.

In a particular embodiment, methods for cryopreserving a biologicalmaterial are provided herein, wherein the at least one IRI compound isan aryl-glycoside, an aryl-aldonamide or a combination thereof.

Preferably, the aryl-glycoside is a para-, ortho- or meta-substitutedaryl-glycoside, and preferably, the para-, ortho- or meta-substitutedaryl-glycoside is a para-, ortho- or meta-substituted aryl-glucosidecorresponding in structure to Formula A reported herein. Preferably, thearyl-glucoside corresponding in structure to Formula A ispara-substituted. Examples of suitable para-substituted aryl-glucosidesfor use in the compositions include, but are not limited to,para-methoxy-phenyl glucoside, para-fluoro-phenyl glucoside andpara-bromo-phenyl glucoside.

Preferably, the aryl-aldonamide is a phenyl-aldonamide corresponding instructure to Formula B reported herein. Examples of suitablephenyl-aldonamides for use in the methods include, but are not limitedto,

In a particular embodiment, methods for cryopreserving a biologicalmaterial are provided herein, wherein the at least one IRI compound ispresent in the solution in a concentration of less than about 400 mM,preferably less than about 200 mM, preferably less than about 100 mM,preferably less than about 10 mM, preferably less than about 1 mM, andpreferably less than about 0.5 mM. In another embodiment, the IRIcompound can be present in the composition in a concentration of about0.5 mM to (and including) about 400 mM, preferably about 55 mM to (andincluding) about 220 mM.

In a particular embodiment, methods for cryopreserving a biologicalmaterial are provided herein, wherein the IRI compound is ann-octyl-D-aldonamide, which is present in a concentration of about 0.5mM or less.

In a particular embodiment, methods for cryopreserving a biologicalmaterial are provided herein, wherein the IRI compound isn-octyl-β-D-galactopyranoside, which is present in a concentration ofabout 22 mM.

In a particular embodiment, methods for cryopreserving a biologicalmaterial are provided herein, wherein the IRI compound is a PMPcarbohydrate derivative.

In a particular embodiment, methods for cryopreserving a biologicalmaterial are provided herein, wherein the PMP carbohydrate derivative ispresent in a concentration of about 110 mM.

In a particular embodiment, methods for cryopreserving a biologicalmaterial are provided herein, wherein the para-, ortho-, ormeta-substituted aryl-glycoside is present in a concentration between(and including) about 55 mM and about 110 mM.

In a particular embodiment, the aryl-aldonomide is present in thecomposition in a concentration between (and including) about 30 mM andabout 200 mM, and preferably about 55 mM.

In a particular embodiment, the biological material is selected from thegroup consisting of an organ, a tissue, and a cell. Examples of a cellinclude, but are not limited to, a cell line, a stem cell, a progenitorcell, a liver cell and a red blood cell, preferably the cell is a redblood cell, a liver cell or a progenitor cell.

Methods for cryopreserving a biological material are also providedherein where the biological material is suspended in cell media.Examples of suitable cell media include Eagle's Minimum Essential Medium(MEM), Dulbecco's Modified Eagle's Medium (DMEM), Roswell Park MemorialInstitute medium (RPMI), Fetal Bovine Serum (FBS), Fetal Calf Serum(FCS), Ham's F-10, Ham's F-12, Hank's buffered salt solution (HBSS),HBSS and dextrose, Medium 199, and a combination thereof. The methodcomprises adding the biological material to a solution comprising atleast one IRI compound, and then cryopreserving the biological materialin cryogenic vials or other suitable container. The vials can be frozenunder rate controlled freezing conditions, such as freezing at 1° C. perminute over 16 hours. The vials can be stored using standardcryopreservation techniques, and then they can be thawed when requiredby removing the vials from the cold storage, and thawing using standardprotocols. Examples of standard cryopreservation techniques includefreezing in liquid nitrogen to about −196° C. and freezing in dry ice toabout −80° C. Examples of standard thawing protocols include ambientthaw or rapid thaw in a water bath between room temperature or 37° C.

In a particular embodiment, methods for cryopreserving a biologicalmaterial are provided herein by suspending the biological material in asolution of at least one IRI compound and additionally at least onecryopreservation agent, wherein the at least one cryopreservation agentis present in amount equal or less than about 30% v/v, preferably equalor less than about 20% v/vof the total solution. In another embodiment,the at least one cryopreservation agent is present in the composition ina concentration of about 0.1% to (and including) about 30% v/v,preferably about 0.1% to (and including) about 20% v/v, preferably about5% to (and including) about 30% v/v, and preferably about 5% to (andincluding) about 20% v/v. Examples of cryopreservation agents includeDMSO, glycerol, polyvinyl alcohol, or combinations thereof.

In a particular embodiment, methods for cryopreserving a biologicalmaterial are provided herein by suspending the biological material in asolution of at least one IRI compound to form a suspension, which iscontained in a vial or other suitable container, wherein the vial isfrozen directly into the storage unit without a rate controlled freezingprotocol.

VI. Methods for Inhibiting Ice Recrystallization in Biological Material

Methods for inhibiting ice recrystallization in a biological materialare provided herein by suspending suspending the biological material ina solution of at least one IRI compound reported herein to form asuspension and cryopreserving the suspension.

Methods for inhibiting ice recrystallization in a biological materialare provided herein, wherein the at least one IRI compound is selectedfrom the group consisting of an alkyl-glycoside, a n-alkyl-aldonamide, an-alkyl-erythronamide, an aryl-glycoside, an aryl-aldonamide, and acombination thereof.

VII. Compositions for Cryopreserving Umbilical Cord Blood

Compositions for cryopreserving umbilical cord blood (UCB) are alsoprovided herein comprising at least one IRI compound reported herein andat least one cryopreservation agent reported herein. As used herein,“umbilical cord blood” refers to whole blood isolated from an umbilicalcord or a fraction of whole blood isolated from an umbilical cord, suchas a fraction comprising hematopoietic stem cells (progenitor cells).

In a particular embodiment, compositions for cryopreserving umbilicalcord blood, such as human umbilical cord blood, are provided hereincomprising at least one IRI compound, wherein the at least one IRIcompound is an aryl-aldonamide, including mono-substituted anddi-substituted aryl-aldonamides.

Preferably, the aryl-aldonamide is a phenyl-aldonamide corresponding instructure to Formula B reported herein or a benzyl-aldonamidecorresponding in structure to Formula B′ reported herein. Examples ofsuitable phenyl- and benzyl-aldonamides for use in the compositioninclude, but are not limited to,

wherein (1a) is phenyl-aldonamide, (2a) isN-(2-fluorophenyl)-D-gluconamide, (3a) isN-(4-methoxyphenyl)-D-gluconamide, (4a) isN-(4-chlorophenyl)-D-gluconamide, and (1b) isN-(2,6-difluorobenzyl)-D-gluconamide.

In a particular embodiment, the at least one IRI compound is present inthe composition in a concentration of less than about 400 mM, preferablyless than about 200 mM, preferably less than about 100 mM, preferablyless than about 10 mM, preferably less than about 1 mM, and preferablyless than about 0.5 mM. In another embodiment, the IRI compound can bepresent in the composition in a concentration of about 0.5 mM to (andincluding) about 400 mM, preferably about 5 mM to (and including) about220 mM.

In a particular embodiment, the phenyl- or benzyl-aldonamide is presentin a concentration between (and including) about 5 mM and about 55 mM.

In a particular embodiment, compositions for cryopreserving umbilicalcord blood are provided herein, wherein the at least onecryopreservation agent is selected from the group consisting of DMSO,glycerol, polyvinyl alcohol, other biopolymers, or a combinationsthereof. The at least one cryopreservation agent is present in thecomposition in a concentration of about 0.1% to (and including) about30% v/v, preferably about 0.1% to (and including) about 20% v/v,preferably about 5% to (and including) about 30% v/v, and preferablyabout 5% to (and including) about 20% v/v.

In a particular embodiment, compositions for cryopreserving umbilicalcord blood are provided herein which further comprise hematopoietic stemcells, white blood cells, or a combination thereof. In anotherembodiment, the umbilical cord blood does not comprise red blood cells.

In a particular embodiment, compositions for cryopreserving umbilicalcord blood are provided herein further comprising a cell medium. Thecell medium may be, for example, saline, dextran, plasma, orcombinations thereof. In various embodiments, saline is included at aconcentration of from about 0.1% to about 2%, or at a concentration ofabout 0.9%, dextran is included at a concentration of from about 1% toabout 10%, or at a concentration of about 5%, and the amount of plasmaincluded varies depending on the final concentrations of cells and othercomponents. In some embodiments, compositions for cryopreservingumbilical cord blood include about 09% saline and 5% dextran, inautoclaved doubly distilled water, an IRI compound and various amountsof plasma.

VIII. Kits for Cryopreserving Umbilical Cord Blood

Kits for cryopreserving umbilical cord blood are provided hereincomprising an IRI compound or composition for cryopreserving umbilicalcord blood as reported herein. The IRI compound and a furthercryopreservation agent, if present, may be in the same composition or inseparation compositions. Additionally, they may be co-packaged forcommon presentation or packaged individually. Instructions can also beprovided in the kit for cryopreservation of umbilical cord blood. Thekits provided herein can further comprise a cell medium. Examples ofsuitable cell medium include saline, dextran, plasma and a combinationthereof.

IX. Methods for Cryopreserving Umbilical Cord Blood

Methods for cryopreserving umbilical cord blood are provided herein byfractionating the umbilical cord blood into a plurality of fractions;including a red blood cell fraction, a buffy coat fraction, includinghematopoietic stem cells and white blood cells, and a serum fraction,isolating the buffy coat fraction, pelleting cells from the buffy coatfraction, suspending the cells in a solution of at least one IRIcompound reported herein to form a suspension, and freezing thesuspension.

In a particular embodiment, methods for cryopreserving umbilical cordblood are provided herein by suspending a blood fraction comprisinghematopoitic stem cells in a solution of at least one IRI compound,wherein the at least one IRI compound is an aryl-aldonamide.

In a particular embodiment, methods for cryopreserving umbilical cordblood are provided herein, wherein the at least one IRI compound is anaryl-glycoside, an aryl-aldonamide or a combination thereof.

Preferably, the aryl-glycoside is a para-, ortho- or meta-substitutedaryl-glycoside, and preferably, the para-, ortho- or meta-substitutedaryl-glycoside is a para-, ortho- or meta-substituted aryl-glucosidecorresponding in structure to Formula A reported herein. Preferably, thearyl-glucoside corresponding in structure to Formula A ispara-substituted. Examples of suitable para-substituted aryl-glucosidesfor use in the compositions include, but are not limited to,para-methoxy-phenyl glucoside, para-fluoro-phenyl glucoside andpara-bromo-phenyl glucoside.

Preferably, the aryl-aldonamide is a mono-substituted phenyl-aldonamidecorresponding in structure to Formula B, a di-substitutedbenzyl-aldonamide corresponding in structure to Formula B′, or acombination thereof. Examples of suitable phenyl- and benzyl-aldonamidesfor use in the methods include, but are not limited to,

wherein (1a) is phenyl-aldonamide, (2a) isN-(2-fluorophenyl)-D-gluconamide, (3a) isN-(4-methoxyphenyl)-D-gluconamide, (4a) isN-(4-chlorophenyl)-D-gluconamide, (5a) isN-(2-chlorophenyl)-D-gluconamide, (6a) isN-(4-Fluorophenyl)-D-gluconamide, (7a) isN-(2-methoxyphenyl)-D-gluconamide, (1b) isN-(2,6-difluorobenzyl)-D-gluconamide, and (2b) isN-(3,5-difluorobenzyl)-D-gluconamide.

In a particular embodiment, methods for cryopreserving umbilical cordblood are provided herein, wherein the at least one IRI compound ispresent in the solution in a concentration of less than about 400 mM,preferably less than about 200 mM, preferably less than about 100 mM,preferably less than about 10 mM, preferably less than about 1 mM, andpreferably less than about 0.5 mM. In another embodiment, the IRIcompound can be present in the composition in a concentration of about0.5 mM to (and including) about 400 mM, preferably about 5 mM to (andincluding) about 220 mM.

In a particular embodiment, the phenyl- or benzyl-aldonamide is presentin a concentration between (and including) about 5 mM and about 55 mM.

Methods for cryopreserving umbilical cord blood are also provided hereinwhere the umbilical cord blood is suspended in a cryopreservationcomposition including an IRI compound. The method comprises adding theumbilical cord blood to a solution comprising at least one IRI compound,and then cryopreserving the umbilical cord blood in cryogenic vials orother suitable container. The vials can be frozen under rate controlledfreezing conditions, such as freezing at 1° C. per minute over 16 hours.The vials can be stored using standard cryopreservation techniques, andthen they can be thawed when required by removing the vials from thecold storage, and thawing using standard protocols. Examples of standardcryopreservation techniques include freezing in liquid nitrogen to about−196° C. and freezing in dry ice to about −80° C. Examples of standardthawing protocols include ambient thaw or rapid thaw in a water bathbetween room temperature or 37° C.

In a particular embodiment, methods for cryopreserving umbilical cordblood are provided herein by suspending the umbilical cord blood in asolution of at least one IRI compound and additionally at least onecryopreservation agent, wherein the at least one cryopreservation agentis present in amount equal or less than about 30% v/v, preferably equalor less than about 20% v/v of the total solution. In another embodiment,the at least one cryopreservation agent is present in the composition ina concentration of about 0.1% to (and including) about 30% v/v,preferably about 0.1% to (and including) about 20% v/v, preferably about5% to (and including) about 30% v/v, and preferably about 5% to (andincluding) about 20% v/v. Examples of cryopreservation agents includeDMSO, glycerol, polyvinyl alcohol, or combinations thereof.

In a particular embodiment, methods for cryopreserving umbilical cordblood are provided herein by suspending the umbilical cord blood in asolution of at least one IRI compound to form a suspension, which iscontained in a vial or other suitable container, wherein the vial isfrozen directly into the storage unit without a rate controlled freezingprotocol.

The methods for cryopreserving umbilical cord blood effectivelycryopreserve hematopoitic stem cells such that upon removing the stemcells from storage, their viability is not substantially decreasedrelative to the viability of stem cells cryopreserved incryopreservatives commonly used in the art and their functionality,i.e., ability to differentiate into colonies, is increased relative tothe functionality of stem cells cryopreserved in cryopreservativescommonly used in the art. In various embodiments, cryopreserving thestem cells with the IRI compounds described herein increases thefunctionality of the stem cells by at least about 1.25 fold, at leastabout 1.5 fold, at least about 2 fold, or at least about 2.5 foldrelative to the functionality of stem cells cryopreserved in only DMSO.

In various aspects, the present technology provides a method forcryopreserving umbilical cord blood, such as, for example, humanumbilical cord blood. The method comprises fractionating whole umbilicalcord blood to generate a fraction comprising hematopoietic stem cells(CD34⁺ cells). In some embodiments, the fraction comprisinghematopoietic stem cells also includes white blood cells (CD45⁺ cells).The method also comprises mixing from about 10,000 to about 100,000, orabout 25,000 to about 75,000, or about 50,000 of the hematopoietc stemcells in a solution comprising at least one ice recrystallizationinhibitor (IRI) compound to form an IRI suspension. Although any IRIcompound described herein can be used, in various embodiments the IRIcompound is at least one unsubstituted aryl-aldonamide ormono-substituted aryl-aldonamide according to Formula B, at least onedi-substituted aryl-aldonamide according to Formula B′, or a combinationthereof. Then, the method comprises freezing the IRI suspension. In someembodiments, the method is performed in cryovials, which are kept on iceuntil ready for placement in a freezer.

In various embodiments, prior to mixing the hematopoietic stem cells ina solution comprising at least one IRI compound, the method furthercomprises transferring the fraction into a cryovial, centrifuging thecryovial to generate a pellet comprising the hematopoietic stem cellsand a supernatant; and removing the supernatant. Removing thesupernatant can be performed by decanting or by aspirating. However,removing the supernatant by aspirating is gentler and disturbs thepellet less than removing the supernatant by decanting. The method alsoincludes adding plasma to the pellet of hematopoietic stem cells to forma suspension comprising the hematopoietic stem cells. The amount ofplasma added to the pellet is dependent on the total volume of cells tobe frozen and on the volume of IRI compound to be added to the cells.Mixing the stem cells in a solution comprising at least one IRI compoundas described above then includes adding the IRI compound to thesuspension comprising the hematopoietic stem cells and mixing togenerate the IRI suspension. The amount of the at least one IRI compoundthat is added to the suspension comprising the hematopoietic stem cellsdepends on the desired final concentration of the IRI compound. Asdescribed above, the at least one IRI compound can be added to a finalconcentration of less than about 400 mM, preferably less than about 200mM, preferably less than about 100 mM, preferably less than about 10 mM,preferably less than about 1 mM, and preferably less than about 0.5 mM.In another embodiment, the IRI compound can be present in thecomposition in a concentration of about 0.5 mM to (and including) about400 mM, preferably about 55 mM to (and including) about 220 mM. Asnon-limiting examples, composition (3a) may be added to a finalconcentration of from about 10 mM to about 55 mM, composition (4a) maybe added to a final concentration of from about 5 mM to about 25 mM,composition (5a) may be added to a final concentration of from about 5mM to about 25 mM, and composition (1b) may be added to a concentrationof from about 10 mM to about 55 mM. However, it is understood thatcompositions (3a), (4a), (5a), and (1b) may be added at concentrationswithin the broader concentrations ranges described herein. In general,the IRI composition is added at a concentration that increasesfunctionality of cryopreserved cells relative to cells stored in theabsence of the IRI compounds and at a concentration that provides a lowcytotoxicity to the cells.

The IRI suspension is then frozen. Freezing the IRI suspension includescooling the IRI suspension at a cooling rate of about −1° C. per minutein a −80° C. freezer for from about 12 to about 48 hours, or for about24 hours to freeze the IRI suspension. The frozen IRI suspension is thentransferred to a liquid nitrogen dewar for storage. Preferably, aftercentrifuging the time until freezing is less than or equal to about 30minutes, less than or equal to about 20 minutes, less than or equal toabout 15 minutes, less than or equal to about 10 minutes, or less thanor equal to about 5 minutes.

X. Method for Inhibiting Ice Recrystallization Umbilical Cord Blood

Methods for inhibiting ice recrystallization in umbilical cord blood areprovided herein by suspending the umbilical cord blood in a solution ofat least one IRI compound reported herein to form a suspension andcryopreserving the suspension.

Methods for inhibiting ice recrystallization in umbilical cord blood areprovided herein, wherein the at least one IRI compound is at least onearyl-aldonamide. In various embodiments, these methods are used tocryopreserve umbilical cord blood.

EXAMPLES

The following examples are merely illustrative, and do not limit thisdisclosure in any way.

Example 1 Ice recrystallization inhibition activity of non-ionicsurfactants n-octyl-β-D-glucopyranoside (compound 3) andn-octyl-β-D-galactopyranoside (compound 4)

n-Octyl-β-D-glycosides are non-ionic surfactants. These surfactants havebeen studied for many applications including crystallization andsolubilisation of membrane proteins as well as lipid-based drug deliverysystems. However, one application which has yet to be explored for thesesurfactants is the ability to inhibit ice recrystallization. Thus, bothcarbohydrate-based non-ionic surfactants n-octyl-β-D-glucopyranoside(compound 3) and n-octyl-β-D-galactopyranoside (compound 4), weresynthesized as detailed in (Capicciotti, C. et al. (2012) “Potentinhibition of ice recrystallization by low molecular weightcarbohydrate-based surfactants and Hydrogelators.” Chem. Sci.3:1408-1416) and their ice recrystallization inhibition activity wasassessed using a “splat cooling” assay.

In this assay, the area of ice crystals are measured after a 30 minuteannealing time at −6.4° C. and compared to a positive control for icerecrystallization resulting in a quantitative measurement of the meanice grain size. For a positive control, a phosphate buffered saline(PBS) solution was utilized. All samples were normalized to the PBSpositive control. The ice recrystallization inhibition activity of thecarbohydrate-based non-ionic surfactants n-octyl-β-D-glucopyranoside(compound 3) and n-octyl-β-D-galactopyranoside (compound 4) is shown inFIG. 1. The ice recrystallization inhibition activity ofn-octyl-β-D-glucopyranoside (compound 3) at 22 mM was identical to thatof D-glucose (Glc), as shown in FIG. 1. This result was consistent withwhat has previously been reported where the nature of the substituent atthe C1 position had little influence on the hydration of themonosaccharide and ice recrystallization inhibition activity. Even at aconcentration of 44 mM, the ice recrystallization inhibition activity ofn-octyl-β-D-glucopyranoside (compound 3) is still identical to that ofthe 22 mM D-glucose. Interestingly, the ice recrystallization inhibitionactivity of its diastereomer, n-octyl-β-D-galactopyranoside (compound4), at 11, 22 and 44 mM was, in all cases, significantly better than 22mM D-galactose (Gal).

Example 2 Ice Recrystallization Inhibition Activity ofCarbohydrate-Based Hydrogelators

N-Octyl-D-aldonamides, compounds 5 and 6 as shown in FIG. 2, are anotherclass of low molecular weight carbohydrate-based compounds known toself-assemble in water. These compounds are prone to aggregation andformation of fibers and hydrogels. Their ice recrystallizationinhibition activity was assessed using a “splat cooling” assay.

In this assay, the area of ice crystals are measured after a 30 minuteannealing time at −6.4° C. and compared to a positive control for icerecrystallization resulting in a quantitative measurement of the meanice grain size. For a positive control, a PBS solution was utilized. Allsamples were normalized to the PBS positive control.

The carbohydrate moiety of the N-octyl-D-aldonamides is in theopen-chain alditol form. Therefore, prior to assessing the icerecrystallization inhibition activity of these hydrogelators, theactivity of D-sorbitol and D-dulcitol and the open-chain reduced form ofD-glucose and D-galactose, respectively, was assessed (as shown in FIG.3). The importance of the pyranose ring for ice recrystallizationinhibition activity is evident as both of these alditols had decreasedactivity relative to the pyranose forms. While hydration numbers for thepyranose forms of D-galactose and D-glucose have previously beenreported, the hydration numbers for the alditols (D-dulcitol andD-sorbitol) are not known. However, it is known that the hydration of amonosaccharide is mostly dependent on the relative stereochemistry ofthe C2 and C4 hydroxyl groups of the pyranose ring. There is asignificant decrease in ice recrystallization inhibition activity forD-dulcitol in comparison to D-galactose, and the ice recrystallizationinhibition activity of both alditols (D-dulcitol and D-sorbitol) isstatistically similar. Without being bound by theory, this resultsuggests that removal of the rigid pyranose ring of a monosaccharidedirectly influences hydration and results in a loss of icerecrystallization inhibition activity.

The ice recrystallization inhibition activity of low molecular weightcarbohydrate-based hydrogelators, compounds 5 and 6, is shown in FIG. 3.Due to the poor solubilities associated with these compounds in waterand PBS, they could only be assessed for ice recrystallizationinhibition activity at 0.5 mM. Surprisingly, N-octyl-D-gluconamide(compound 5, NOGlc) exhibited potent ice recrystallization inhibitionactivity at 0.5 mM, while its diastereomer N-octyl-D-galactonamide(compound 6, NOGal) exhibited only moderate ice recrystallizationinhibition activity. In fact, the activity of compound 6 at 0.5 mM wasstatistically similar to the activity of D-galactose at 22 mM. Theseresults are highly unprecedented and are the first example where aD-glucose derivative was a more potent inhibitor of icerecrystallization than a D-galactose derivative. Furthermore,N-octyl-D-gluconamide (compound 5) is the first example of a smallmolecule having potent ice recrystallization inhibition activity at asignificantly lower concentration than 22 mM.

Interestingly, compound 5 exhibited potent ice recrystallizationinhibition activity at 0.5 mM, but compound 7 exhibited only moderateice recrystallization inhibition activity at a higher concentration of22 mM. Compound 8 exhibited similar ice recrystallization inhibitionactivity to compound 7 at 22 mM further validating the importance of theproton on the amide bond in 5 for potent ice recrystallizationinhibition activity. In addition, replacement of the amide bond inD-glucose derivative 5 with an ether linkage (9) resulted in a dramaticdecrease in ice recrystallization inhibition activity. Ether-linkedcompound 9 exhibited ice recrystallization inhibition activity similarto compounds 7 and 8 at 22 mM. Collectively, these results indicate thatthe amide bond of compound 5 is crucial for potent ice recrystallizationinhibition activity.

Interestingly, compound 9 is the open-chain alditol derivative of thenon-ionic D-glucose-based surfactant compound 3 and compound 9 was abetter inhibitor of ice recrystallization than compound 3 at 22 mM(FIGS. 1 and 3). This was surprising as the ice recrystallizationinhibition activity for alditols D-sorbitol and D-dulcitol (FIG. 3) wasdecreased in comparison to the corresponding pyranose forms of thesugars (D-glucose and D-galactose, respectively). As non-ionicD-galactose surfactant 4 exhibited potent ice recrystallizationinhibition activity at 22 mM (FIG. 1), we synthesized compound 10 tofurther examine the influence of the alditol moiety on icerecrystallization inhibition activity. Compound 10 is the open-chainalditol derivative of compound 4. Unfortunately, even at 11 mM, compound10 was insoluble in PBS, and thus its ice recrystallization inhibitionactivity could not be assessed at similar concentrations which compounds4 and 9 were assessed.

Example 3 Interactions with the Ice Lattice

It has previously been demonstrated that C-linked AFGP analogues 1 and 2exhibited potent ice recrystallization inhibition activity but did notpossess TH activity, which suggested that the mechanism of action forthese potent inhibitors of ice recrystallization is different fromnative AF(G)Ps as no direct interaction with the ice lattice wasobserved. As both compound 4 (at 22 mM) and compound 5 (at 0.5 mM) arenovel potent low molecular weight inhibitors of ice recrystallization,whether they were interacting directly with the ice lattice wasinvestigated. Carbohydrate derivatives (compounds 3-6) were examined forTH activity using a Clifton Nanoliter Osmometer. See Tam, R. et al.(2009) “Solution Conformation of C-Linked Antifreeze GlycoproteinAnalogues and Modulation of Ice Recrystallization” J. Am. Chem. Soc.131:15747-15753; Tam, R. et al. (2008) “Hydration Index—A BetterParameter for Explaining Small Molecule Hydration in Inhibition of IceRecrystallization” J. Am. Chem. Soc. 130: 17494-17501.

In this assay a single droplet of the sample in water was suspended inthe center of an oil-filled well in a sample holder plate. The samplewas then rapidly frozen using a thermoelectrically controlled microscopestage, then the temperature was raised and the sample melted until onlya single ice crystal remained. Once a single ice crystal was obtained,its growth and behavior upon increasing/decreasing the temperature wasviewed under a microscope.

The above synthesized C-linked AFGP analogues and carbohydratederivatives were assessed for TH activity at 10 mg/mL. Compounds 3-6could not be assessed for TH activity at this concentration due to theiramphiphilic nature. During the process of melting the frozen sample toobtain a single ice crystal, the droplet dissolved in the oil resultingin one phase. Thus, compounds 3 and 4 were assayed at a concentration of1 mg/mL, and compounds 5 and 6 were assayed at a concentration of 0.01mg/mL. None of these derivatives (compounds 3-6) exhibited TH activityor dynamic ice shaping at these concentrations as shown in FIGS. 4(a)-(d). The absence of dynamic ice shaping indicated there was nointeraction with the ice lattice.

Many AF(G)Ps have measureable TH gaps at concentrations as low as 0.5mg/mL. However, only hyperactive AFPs have been reported to havemeasureable TH activity at concentrations of 0.01-0.05 mg/mL. Therefore,native AFGP-8 was assayed at a concentration of 0.01 mg/mL for dynamicice shaping and TH activity. While no measureable TH gap was obtainedfor AFGP-8 at 0.01 mg/mL, dynamic ice shaping was observed as shown FIG.4 e. This indicated that even at a low concentration of 0.01 mg/mLevidence of an interaction with the ice lattice can still be observed,even in the absence of a measureable TH gap. The fact that compounds 5and 6 do not exhibit this activity suggests that these compounds are notinteracting with the ice lattice. However, as compounds 5 and 6 couldnot be tested for TH activity at a concentration higher than 0.01 mg/mL,solid-state NMR was used to investigate interactions with the icelattice.

Example 4 Solid-State NMR

Solid-state NMR has been utilized to observe the interaction of a typeIII AFP with ice. In this study, one of the findings was that the ²Hspin-lattice relaxation rate (R₁) of frozen D₂O in the presence of theAFP was much faster in comparison to the ²H R₁ of frozen D₂O on its own.It was also found that the R₁ of frozen D₂O in the presence of ubiquitinwas similar to that of frozen D₂O on its own. The faster relaxation inthe presence of the AFP was hypothesized to be a direct result of theAFP binding to ice as the R₁ of frozen D₂O in the presence of thenegative control, ubiquitin, was similar to that of D₂O on its own, andubiquitin does not interact with the ice lattice.

Prior to measuring the ²H R₁ of frozen D₂O in the presence of compounds3-6, the R₁ of pure D₂O at −25° C., as well as the R₁ of frozen D₂O inthe presence of three positive controls and one negative control for icebinding was measured. The saturation recovery curves for thesemeasurements are shown in FIG. 5 a. The ²H relaxation rate for frozenD₂O was R₁=0.0154±0.0002 s⁻¹. Two of the positive controls chosen were atype I AFP (winter flounder, Pseudopleuronectes americanus) and a typeIII AFP (ocean pout, Macrozoarces americanus). As can been seen fromFIG. 5 a, the R₁ of frozen D₂O with both type I (R₁=0.13±0.01 s⁻¹) andtype III AFP (R₁=0.22±0.01 s⁻¹) is much faster than the R₁ of frozen D₂Oon its own. The third positive control used was the wild-type AFP of aperennial ryegrass, Lolium perenne (WT LpAFP) (R₁=0.039±0.002 s⁻¹). ThisAFP was chosen as it can be recombinantly expressed in Escherichia coli(E. coli). In addition, a mutant of this AFP (T67Y LpAFP) could beutilized as a negative control for ice binding as it has previously beendemonstrated that substitution to a bulky tyrosine residue disrupted thediscrete complementarity between the ice-binding face of the AFP andice, resulting in minimal TH activity. Thus, WT LpAFP and T67Y LpAFPwere recombinantly expressed in E. coli (Yu, S. et al. (2010) “Icerestructuring inhibition activities in antifreeze proteins with distinctdifferences in thermal hysteresis.” Cryobiology 61(3):327-334), and theR₁ of frozen D₂O in their presence was measured (FIG. 5 a). The R₁ offrozen D₂O with T67Y LpAFP (R₁=0.018±0.001 s⁻¹) was similar to that offrozen D₂O on its own. In contrast, a significantly faster ²H relaxationrate was observed in the presence of all three positive controls whichare known to bind to ice.

Next, the saturation recovery curves of frozen D₂O in the presence ofcompounds 3-6, as well as D-glucose and D-galactose, were recorded. Allof these molecules are significantly smaller than the AFP positivecontrols. Therefore, to account for the drastic difference in molecularweights, the concentration which each carbohydrate derivative wasmeasured was corrected to a total overall proton concentration of 1234mM, the overall protein proton concentration used for the Type III AFPin the original study. As shown in FIG. 5( b), in the presence ofD-galactose derivative, compound 4, the R₁ of frozen D₂O(R₁=0.0209±0.0002 s⁻¹) was similar to that of D₂O on its own, suggestingit is not interacting with the ice lattice. Similar results were alsoobtained for D-galactose, D-glucose, and compound 3, as shown in FIGS.14 (a)-(c), where the R₁ of frozen D₂O in the presence of thesecompounds (R₁=0.0154±0.0003 s⁻¹; R1=0.0132±0.0001 s⁻¹; R₁=0.0190±0.0002s⁻¹) was similar to the R₁ of frozen D₂O on its own. These resultsfurther validated the previous TH results and suggest that the potentice recrystallization inhibition activity of compound 4 at 22 mM is notdue to an interaction with the ice lattice.

The saturation recovery curve of frozen D₂O with compound 5 is shown inFIG. 5( c). Surprisingly, the R₁ of frozen D₂O in the presence ofcompound 5 (R₁=0.0310±0.0005 s⁻¹) was faster than that of frozen D₂O onits own. However, since the TH measurements of compound 5 indicated nodynamic ice shaping, it seemed that the faster R₁ was not due to aninteraction with the ice lattice. As the concentration of the lowmolecular weight carbohydrate derivatives were corrected to a totaloverall proton concentration of 1234 mM, this corresponded to a 42 mMconcentration of compound 5 in solution. At 42 mM, compound 5 forms ahydrogel, and so consequently the NMR measurements were conducted on afrozen hydrogel. The formation of a gel is inevitably going to beaccompanied by a decrease in correlation time due to an increasedmobility of the water molecules in the solid-state. This is expected tocause an increase in R₁, which was seen experimentally. Therefore,without being bound by theory, it is believed that the faster R₁observed for frozen D₂O with compound 5 is due to increased molecularmotion during these measurements caused by gelation, which was furthersupported by the saturation recovery curve of frozen D₂O withD-galactose derivative (compound 6) (FIG. 5( d)). Due to solubilityproblems, the highest concentration at which compound 6 could bemeasured was its gelation concentration of 0.5% w/v, which correspondsto a concentration of 16 mM. A similar effect to what was seen withcompound 5 (FIG. 5( c)) was observed with compound 6 where the R₁ of D₂Oin the frozen hydrogel of compound 6 (R₁=0.030±0.001 s⁻¹) was fasterthan the R₁ of frozen D₂O on its own (FIG. 5( d)). However, a 16 mMsolution of compound 6 has a total overall proton concentration of only479 mM and at a similar protein proton concentration with the WT LpAFP,the measured R₁ value (R₁=0.0133±0.0005 s⁻¹) is similar to frozen D₂O onits own (FIG. 5( d)). As expected, the effect of ice binding on R₁ isconcentration dependent. Thus, at the lower total proton concentration afaster R₁ was observed for the frozen hydrogel of compound 6. Theseresults suggest that the faster relaxation rates of frozen D₂O observedwith compounds 5 and 6 are likely due to a gelation effect.

Interestingly, the R₁ values of frozen D₂O in both of the hydrogels(compounds 5 and 6) are similar (FIG. 5( c)-(d)) despite their dramaticdifference in ice recrystallization inhibition activity. This resultfurther validates the hypothesis that simply the ability to form ahydrogel does not necessarily result in potent ice recrystallizationinhibition activity. Regardless of concentration, both compounds 5 and 6were studied as frozen hydrogels in the solid-state NMR experiments, andboth had the same relative increase in the relaxation rate of frozenD₂O. However, only D-glucose derivative, compound 5, had potent icerecrystallization inhibition activity (FIG. 3).

Example 5 Human Liver Cell Viability

Human liver cell viability in PMP-glucoside (compound 17, PMP-Glc) andN-octyl-D-gluconamide (compound 5, NOGlc) was assessed using a MTTAssay.

In the assay, HepG2 cells (human hepatocellular carcinoma cells) wereharvested and incubated at 37° C. overnight in a 96-well plate to aconcentration of 20,000 cells per well using Minimum Essential Medium(MEM) as media. The following day (or once cells were confluent) themedia was removed and cells were incubated at 37° C. overnight with MEMsupplemented with desired concentration of PMP-Glc and NOGlc (100 μL ofsolution added). The following day the plates were centrifuged (700 RPMfor 3 minutes) and the solutions were removed. 200 μL of MEM was addedto each well, followed by 50 μL of 2.5 mg/mL MTT solution in Hank'sBalanced Salt Solution (HBSS) to a final concentration of 0.5 mg/mL MTTper well. Then the plates were incubated for 3 hours at 37° C. Afterincubation, the plates were centrifuged (700 RPM for 3 minutes) and 200μL of a 10% TritonX/0.1 N HCl in isopropanol solution was added to eachwell. The plates were incubated in the dark for 1 hour, then shaken andthe absorbance at 570 nM of each well was read. % viability at 37° C. ofPMP-Glc at a concentration 11 mM, 22 mM, 44 mM, 110 mM and 220 mMPMP-Glc was measured, and the results are shown in FIG. 12. % viabilityat 37° C. of NOGlc at a concentration of 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2mM and 5 mM was measured. The results are shown in FIG. 13. Also, %viability at 37° C. of PMP-Glc, pFPh-Glc and pBrPh-Glc at aconcentration of 11 mM, 22 mM, 44 mM, 55 mM, 110 mM and 220 mM wasmeasured under the same conditions for the MTT Assay described above.The results are shown in FIG. 25.

Example 6 Red Blood Cell Cryopreservation

The cryopreservation ability of various carbohydrates and carbohydratederivatives with and without the addition of glycerol as cryoprotectantswith red blood cells (RBCs) obtained from peripheral blood was assessed.Hematocrits and % hemolysis (obtained by Drabkin's assay) were analyzedfor non-nucleated samples and post-thaw samples that were frozen underslow and rapid cooling conditions. A summary of the cryoprotectantsolutions with the freezing temperatures analyzed is shown in Table 1below.

Fresh whole blood was obtained then centrifuged at 1000 g for 10 minutesat 4° C. The supernatant was then removed, which included plasma,platelets and white blood cells. The RBCs were then washed with a salinesolution and then centrifuged to concentrate the cells. ConcentratedRBCs were then split into 1.5 mL Eppendorf tubes, with 350 μL of RBCsadded to each (1 Eppendorf tube per cryo-solution for each freezingtemperature analyzed). 350 μL of desired cryoprotectant was added toeach tube. The cells were then incubated at room temperature or 0° C.for 10 minutes, then 300 μL of RBC/cryo-solution mixture was transferredto cryogenic tubes, with each sample being run in duplicate (300 μL×2for each cryosolution and each freezing temperature).

The cryogenic tubes were then placed in a pre-cooled ethylene glycolbath at −5° C. and were held at this temperature for 5 minutes. After 5minutes, duplicate non-nucleated samples for each cryo-solution wereremoved from the bath (−5° C. non-nuc for each cryo-solution). Thesamples remaining in the bath were then nucleated and held at −5° C. for5 minutes. After this time, duplicate samples for each cryo-solutionwere removed and immediately thawed and analyzed for % hemolysis (−5° C.slow), and duplicate samples for each cryo-solution were placed inliquid nitrogen or packed in dry ice then placed in −80° C. freezer (−5°C. rapid, to either −196° C. (liq. N₂) or −80° C. (dry ice)).

The samples remaining in the bath were then cooled at a rate of 1° C.Samples were removed at −15° C. and −40° C. and were either immediatelythawed and analyzed for % hemolysis (−15° C. slow and −40° C. slow) orwere placed in liquid nitrogen or packed in dry ice then placed in −80°C. freezer (−15° C. rapid and −40° C. rapid, to either −196° C. (liq.N₂) or −80° C. (dry ice)). Samples done under rapid cooling conditionsremained in liquid nitrogen or −80° C. freezer for a minimum time of 30minutes then were thawed and analyzed for % hemolysis.

For cyroprotectant solutions 1-23 in Table 1, rapid conditions used wereplacing samples into liquid nitrogen. For cryoprotectant solutions24-31, rapid conditions used were placing samples into dry ice for 5minutes then placing in −80° C. freezer.

In cryoprotectant solutions 5-8, OGG-Gal refers to analog 1 describedabove. Each cryoprotectant solution was tested at each correspondingfreezing temperature listed. For example, cryoprotectant solution 1,(i.e. RBCs+2 mM NOGlc+20% glycerol) was tested at −5° C. non-nuc, −5° C.slow, −5° C. rapid, −15° C. slow and −15° C. rapid.

TABLE 1 Cryoprotectant Solutions Freezing Temperatures 1) RBCs + 2 mMNOGlc + 20% glycerol,  −5° C. non-nuc 2) RBCs + 2 mM NOGlc,  −5° C. slow3) RBCs + 5 mM NOGlc + 20% glycerol,  −5° C. rapid 4) RBCs + 5 mM NOGlc−15° C. slow −15° C. rapid 5) RBCs + 2 mg/mL OGG-Gal + 20% glycerol, −5° C. non-nuc 6) RBCs + 2 mg/mL OGG-Gal,  −5° C. slow 7) RBCs + 5mg/mL OGG-Gal + 20% glycerol,  −5° C. rapid 8) RBCs + 5 mg/mL OGG-Gal−40° C. slow −40° C. rapid 9) RBCs + 220 mM Sucrose + 20% glycerol,  −5°C. non-nuc 10) RBCs + 220 mM Sucrose,  −5° C. slow 11) RBCs + 500 mMSucrose + 20% glycerol,  −5° C. rapid 12) RBCs + 500 mM Sucrose, −15° C.slow 13) RBCs + 220 mM Fructose + 20% glycerol, −15° C. rapid 14) RBCs +220 mM Fructose, −40° C. slow 15) RBCs + 500 mM Fructose + 20% glycerol,−40° C. rapid 16) RBCs + 500 mM Fructose, 17) RBCs + 110 mM PMP-Glc +20% glycerol, 18) RBCs + 110 mM PMP-Glc, 19) RBCs + 220 mM PMP-Glc + 20%glycerol, 20) RBCs + 220 mM PMP-Glc, 21) RBCs + 20% glycerol + 110 mMPMP-Glc (incubation at room temperature or 0° C.), 22) RBCs + 20%glycerol (incubation at room temperature or 0° C.), 23) RBCs + 20%glycerol + 220 mM PMP-Glc (incubation at room temperature or 0° C.) 24)RBCs + 5% glycerol + 110 mM PMP-Glc, 25) RBCs + 10% glycerol + 110 mMPMP-Glc, 26) RBCs + 15% glycerol + 110 mM PMP-Glc, 27) RBCs + 20%glycerol + 110 mM PMP-Glc, 28) RBCs + 5% glycerol, 29) RBCs + 10%glycerol, 30) RBCs + 15% glycerol, 31) RBCs + 20% glycerol,

The results showing cryoprotection ability of the cryoprotectantsolution of PMP-Glc with a range of concentrations of glycerol is shownin FIGS. 15-18. The results showing cryoprotection ability of thecryoprotectant solution of sucrose is shown in FIG. 19. The resultsshowing cryoprotection ability of the cryoprotectant solution offructose is shown in FIG. 20. The results showing cryoprotection abilityof the cryoprotectant solution of OGG-Gal is shown in FIG. 21. Theresults showing cryoprotection ability of the cryoprotectant solution of110 mM PMP-Glc is shown in parallel to cryoprotectant solutions of 110mM pFPh-Glc and 55 mM pBrPh-Glc under different sets of conditions inFIGS. 22 and 23.

In the experiments summarized in FIGS. 22 and 23, two separate cryovialscontaining the RBCs were suspended in the cryoprotectant solution andplaced in a cryobath where the temperature was decreased at precisely 1°C./minute. At −5° C., each sample was nucleated to ensure ice formationand the temperature was decreased at −1° C./minute until the desiredtemp was reached (−5, −15, −40 or −50). At this temperature, one of thecryovials was removed and thawed rapidly in a bath set to 37° C. Theother sample was placed in a −80° C. freezer and stored for 1 hour. Thesample was then thawed rapidly.

Example 7 Cryopreservation of Tfl-α (or Tfl-a) Cells with (i) DMSO Aloneand (ii) Aryl-Aldonamides (Compounds 1a, 2a and 3s) with and withoutDMSO

Tfl-α cells were cultured to a final concentration of 2-3×106 cells/mL.Three million cells were transferred into cryovials and media wereremoved by aspiration after centrifugation at 1000 rpm for five minutes.100 μL of RPMI media (10% FBS) supplemented with the cryopreservative ofinterest was added to the cryovial and vortexed. The cryovials werecooled at a rate of −1° C./min for 24 hours to −80° C. before beingtransferred to a liquid nitrogen dewar for storage until analysis.

Viability/Apoptosis Analysis

Cryovials were thawed under fast thaw conditions (submerged in 37° C.water bath). The cells were then diluted to a concentration of 3×10⁶cells/mL with 1× Annexin V Binding Buffer and a cell count was obtainedby hemacytometer (to obtain cell recovery). A 400 μL aliquot wastransferred to a flow cytommetry tube. 10 μL of each Annexin V FITC (forapoptosis) and 7-AAD (for viability) dyes were added, the tube wasvortexed and allowed to incubate in the dark for 15 minutes. Thesolution was diluted to 1 mL with 1× Annexin V Binding Buffer, vortexedand analyzed by flow cytommetry. FIG. 26( a) shows the viability of thecells stored at −196° C. for 48 hours without a cryoprotectant solution,but only with 0%, 2% or 10% DMSO before flow cytometry analysis. With 2%DMSO, cell viability reached about 45%, and with 10% DMSO, cellviability reached around 78%.

FIG. 26( b) shows Tfl-α cell viability when the cells were treated witha cryoprotectant solution containing an aryl-aldonamides of Formula B(compounds 1a, 2a and 3a) at 55 mM with 0%, 2% or 10% DMSO. The resultsare shown in Table 2.

TABLE 2 Cell viability and apoptosis of TF1-α cells Aryl-aldonamides inthe cryoprotectant Cell Viability solution (55 mM) % DMSO (%) Apoptosis(%)

0 2 10   0.07 34.71 68.82 0   0.9  4.22

0  1.27 0   2 81.28 1.5  10  92.83 3.37

0 2 10   0.16 74.33 95.33 0   1.38 2.73

indicates data missing or illegible when filed

Example 8 Cryopreservation of Umbilical Cord Blood Synthesis andCharacterization of N-(4-chlorophenyl)-D-gluconamide (5a)

The synthesis of (5a) is shown in FIG. 27( a). To a solution ofD-gluconic acid-d-lactone (0.20 g, 1.12 mmol) in acetic acid (5 mL) wasadded 4-chloroaniline (0.12 mL, 1.12 mmol). The mixture was stirredunder reflux for 2 hours. The crude product was precipitated withhexanes, filtered and crude solid was recrystallized in EtOH to afford(5a) as white crystals (180 mg, 52%). ¹H NMR (400 MHz, DMSO-d₆): δ 9.7(s, 1H), 7.8 (d, J=9.1 Hz, 2H), 7.35 (d, J=8.8 Hz, 2H), 5.71 (d, J=5.3Hz, 1H), 4.59 (d, J=4.9 Hz, 1H), 4.55-4.53 (m, 2H), 4.36 (t, J=5.7 Hz,1H), 4.18 (dd, J=5.1, 3.7 Hz, 1H), 4.02-3.99 (m, 1H), 3.61-3.57 (m, 1H),3.52-3.51 (m, 2H), 3.42-3.36 (m, 1H). ¹³C NMR (400 MHz, DMSO-d₆): δ171.77, 137.56, 128.40, 126.89, 121.17, 74.20, 72.19, 71.53, 70.31,63.27. LRMS (ESI): m/z calcd. for C₁₂H₁₆ClNaNO₆ [M+Na]⁺328.70. found327.95.

Synthesis and Characterization of N-(2,6-difluorobenzyl)-D-gluconamide(1b)

The synthesis of (1b) is shown in FIG. 27( b). To a solution ofD-gluconic acid-d-lactone (0.20 g, 1.12 mmol) in MeOH (15 mL) was added2,6-difluoroaniline (0.13 mL, 1.12 mmol). The mixture was stirred underreflux for 24 hours. The solvent was evaporated and the residue wasrecrystallized in EtOH to afford (1b) as white crystals (89%). ¹H NMR(300 MHz, DMSO-d₆): δ 7.89 (t, J=5.73 Hz, 1H), 7.33-7.43 (m, 1H), 7.07(t, J=8.09 Hz, 2H), 5.35 (d, J=5.32 Hz, 1H), 4.53 (d, J=5.00 Hz, 1H),4.38-4.48 (m, 3H), 4.27-4.34 (m, 1H), 4.00 (dd, J=5.18 Hz, 3.77 Hz, 1H),3.87-3.91 (m, 1H), 3.53-3.59 (m, 1H), 3.44-3.45 (m, 2H), 3.35-3.39 (m,1H). ¹³C NMR (400 MHz, DMSO-d₆): δ 172.64, 167.72 (d, J_(C,F)=8.41 Hz),160.26 (d, J_(C,F)=8.18 Hz), 130.26 (t, J_(C,F)=10.37 Hz), 114.67 (t,J_(C,F)=19.56 Hz), 111.96 (dd, J_(C,F)=18.85 Hz, 6.51 Hz), 74.02, 72.81,71.98, 70.56, 63.83. ¹⁹F NMR (300 MHz, DMSO-d₆): δ −114.5. LRMS (ESI):m/z calcd. for C₁₃H₁₇F₂KNO₆ [M+K]⁺ 360.07. found 360.09.

Ice Recrystallization Inhibition Activity of Aryl-Alditols

N-aryl-D-gluconamides (3a), (2a), (4a), and (1b) are a class of lowmolecular weight carbohydrate-based compounds. The ice recrystallizationinhibition activity of (3a), (2a), (4a), and (1b) was assessed using a“splat cooling” assay. In this assay, the area of ice crystals aremeasured after a 30 minutes annealing time at −6.4° C. and compared to apositive control for ice recrystallization resulting in a quantitativemeasurement of the mean ice grain size. For a positive control, PBS wasutilized. All samples were normalized to the PBS positive control. TheIRI activity of (3a), (2a), (4a), and (1b) is shown in FIG. 28. As shownin FIG. 28, each of (3a), (2a), (4a), and (1b) resulted in ice crystalswith a mean grain size much smaller than that of ice crystals generatedin PBS alone. More specifically, (3a) resulted in about a 95% decreasein grain size, (2a) resulted in about a 95% decrease in grain size, (4a)resulted in about a 60% decrease in grain size, and (1b) resulted inabout 85% decrease in grain size.

Processing of Human Umbilical Cord Blood (UCB)

The processing of UCB procedure was based upon Rubinstein's method forUCB processing (Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 10119-10122),which is incorporated herein by reference.

Whole umbilical cord blood (UCB) should be stored at room temperature(15-25° C.) and processed within 48 hrs of collection. UCB is removedfrom collection bag and spilt in equal parts into 50 mL Falcon tubes. Nomore than 25 mL of UCB should be added per Falcon tube. UCB is dilutedwith 6% hetastarch (Hespan) to obtain a final concentration of 1%hetastarch and incubated for 10 minutes at room temperature. The tubesare then centrifuged at 50 g at 10° C. for 15-20 minutes, depending onvolume. The centrifuge should not brake to stop. For total volumes of 30mL, 15-17 minute centrifugation is utilized and for total volumes of 35mL, 17-20 minute centrifugation is utilized. The supernatant and buffycoat (plasma and leukocytes) are removed carefully (not collecting RBCs)from the tubes and collected into new 50 mL Falcon tubes. These tubesare centrifuged at 400 g and 10° C. for 10 mins to pellet cells. Thecentrifuge brake can now be utilized. Plasma is removed and kept in new50 mL Falcon tubes on ice. The cell pellets resuspended and combined inplasma to a total volume of 20 mL to generate leukocyte concentrate(LC). The LC is kept on ice for the duration of its use. Totalmononuclear (CD45⁺) and CD34⁺ (hematopoetic stem cells) cellconcentrations and viability are determined by flow cytometry (see FlowCytometry procedure below). Aliquots of LC (50,000 CD34⁺ cells) areutilized for cryopreservation experiments (see CryopreservationExperiments procedure below).

Cryopreservation Experiments

Aliquots of LC (50,000 CD34⁺ cells) are added to 2.0 mL cryovials. Foreach experiment, four cryovials are prepared (two for post-thawviability analysis using flow cytometry and two for functionalityanalysis using the colony forming unit assay). Cryovials are kept on iceuntil placement in −80° C. freezer. Cryovials are centrifuged at 400 gand 10° C. for 5 minutes to pellet cells. After centrifugation, the timetaken until placement in −80° C. freezer should not exceed 10 minutes.Supernatant is removed by aspiration or by decanting, but preferably byaspiration. Plasma (volume depends on desired cryosolutionconcentration) is added and cells are suspended by pipetting. Pre-madecryosolutions (see Cryosolutions) are added (volume depends on desiredcryosolution concentration) and mixed by pipetting. Cryovials are placedin a Mr. Frosty® rate controlled freezing container which is then placedin a −80° C. freezer for 24 hours. By placing the cryovials in thefreezing container, their contents undergo a cooling rate of about −1°C. per minute. After 24 hours, the cryovials are transferred to a liquidnitrogen dewar for storage until thawed for analysis.

Cryosolutions

Cryosolutions are prepared in distilled autoclaved water supplementedwith 0.9% saline and 5% dextran for cryopreservation experiments anddiluted with different volumes of plasma in cryovials depending ondesired concentration of ice recrystallization inhibitor.N-(4-chlorophenyl)-D-gluconamide (4a) andN-(2-fluorophenyl)-D-gluconamide (2a) are prepared at 25 mM andN-(4-methoxyphenyl)-D-gluconamide (3a) andN-(2,6-difluorobenzyl)-D-gluconamide (1b) are prepared at 55 mM. 100,50, 33 or 20 μL of 25 mM N-(4-chlorophenyl)-D-gluconamide (4a) orN-(2-fluorophenyl)-D-gluconamide (2a) are added to cryovials containing50,000 CD34⁺ cells suspended in 0, 50, 67, or 80 μL of plasma,respectively, for a final IRI concentration of 25, 12.5, 8 or 5 mM,respectively. 100, 50, 33 or 20 μL of 55 mMN-(4-methoxyphenyl)-D-gluconamide (3a) orN-(2,6-difluorobenzyl)-D-gluconamide (1b) are added to cryovialscontaining 50,000 CD34⁺ cells suspended in 0, 50, 67, or 80 μL ofplasma, respectively, for a final IRI concentration of 55, 22.5, 18 or11 mM, respectively.

Flow Cytometry

Total mononuclear cell (CD45+) and progenitor cell (CD34+) viability isassessed by flow cytometry using International Society of Hematotherapyand Graft Engineering (ISHAGE) guidelines (J. Hematother. Stem Cell Res.1996, 5, 213-226). Fluorescently tagged antibodies for both CD45 andCD34 are employed to identify the population within the leukocytes whichare hematopoietic stem cells (HSCs) using the ISHAGE gating strategy.HSCs are identified as being both CD45⁺ and CD34⁺, by employing7-aminoactinomycin D (7-AAD) in the analysis, cell viabilities of bothtotal mononuclear cells and HSCs can be quantified. Countbright®counting beads are employed to obtain cell concentrations and thus cellrecovery post-thaw can be quantified.

Freshly Obtained UCB

For fresh UCB, flow cytometry analysis is performed within 48 hours ofUCB collection. 100 uL of the LC is diluted with 900 uL Dulbecco'sphosphate buffered saline (DPBS). 200 uL of this cell suspension isadded to a polypropylene flow cytometry tube. 8 uL of each CD45fluorescein isothiocyanate (FITC) and CD34 phycoerythrin (PE) are addedand incubated in the dark at room temperature for ten minutes. 8 uL of7-AAD is added and cell suspension is allowed to incubate for anadditional five minutes in the dark at room temperature. 20 uL ofCountbright® counting beads are added and the suspension is diluted to 1mL with 1× red blood cell lysis buffer. Samples are analyzed using aBeckman Coulter Gallios Flow Cytometer.

Post-Thaw Viability Analysis

For frozen UCB, flow cytometry analysis is performed within 1 hour ofthawing samples in a 37° C. water bath. After thawing samples in a 37°C. water bath, the cell suspension is diluted with 900 μL of DPBS. 200uL of this cell suspension is added to a polypropylene flow cytometrytube. 8 uL of each CD45 fluorescein isothiocyanate (FITC) and CD34phycoerythrin (PE) are added and incubated in the dark at roomtemperature for ten minutes. 8 uL of 7-AAD is added and cell suspensionis allowed to incubate for an additional five minutes in the dark atroom temperature. 20 uL of Countbright® counting beads are added and thesuspension is diluted to 1 mL with 1×red blood cell lysis buffer.Samples are analyzed using a Beckman Coulter Gallios Flow Cytometer. %viability post-thaw of (3a) at 11 mM, 18 mM, 27.5 mM, and 55 mM wasmeasured and the results are shown in FIG. 29. % viability post-thaw of(2a) at 5 mM, 8 mM, 12.5 mM and 25 mM was measured and the results areshown in FIG. 30. % viability post-thaw of (1b) at 11 mM, 18 mM, 27.5mM, and 55 mM was measured and the results are shown in FIG. 31. %viability post-thaw of (4a) at 5 mM, 8 mM, 12.5 mM and 25 mM wasmeasured and the results are shown in FIG. 32. These results show thatthe cryopreservatives (3a), (2a), (4a), and (1b) result in CD34+ cellviability that is substantially similar to the control that contained10% DMSO only. In some instances, the phenyl-aldonamides resulted inhigher viabilities than the DMSO control. Overall, (3a), (2a), (4a), and(1b) resulted in viabilities of from about 60% to about 95%.

Colony Forming Unit (CFU) Assay

Cryovials are thawed in a 37° C. water bath and diluted with 900 μL ofIMDM (10% FBS, 1% penicillin/streptomycin). Cryovials are placed on iceuntil washed. Samples are mixed by pipetting and 80 μL is transferred toa 15 mL Falcon tube. 5 mL of Iscove's Modified Dulbecco's Medium (IMDM)supplemented with 10% fetal bovine serum (FBS) is added and this mixtureis centrifuged at 1100 rpm for six minutes to yield a cell pellet and asupernatant. The supernatant is removed by aspiration and the cellpellet is resuspended in 1 mL IMDM (2% FBS) to form a suspension. Thesuspension is mixed by pipetting. 150 μL of this cell suspension isadded to 3 mL Methocult™ media and plating is performed in accordancewith manufacturer's instruction. The suspension with Methocult™ media ismixed adequately by vortexing for 20 seconds. The contents are allowedto settle and bubbles are allowed to subside before plating(approximately 5-10 minutes). Using a 3 mL syringe and 16 gauge bluntneedles, 2.5 mL of the cell suspension in Methocult™ is taken from thebottom. 1 mL of this 2.5 mL is added to two 5 mL cell culture dishes.The dishes are swirled to ensure cells in Methocult™ medium coat theentire bottom of the dish. A third 5 mL dish is prepared to containdistilled autoclaved water without a lid. These three 5 mL dishes areplaced in a 100 mL plate; two 5 mL dishes contain 1 mL of cellsuspension in Methocult™ medium with the lid on and one containingdistilled autoclaved water with the lid off. The lid is placed on the100 mL plate and stored in a 37° C. incubator (5% CO₂) for two weeks.

After two weeks of incubation, colonies which have formed are countedand scored according to Standardized Guide provided by StemCell™Technologies, Inc. Individual and total colonies formed for (3a) at 11mM, 18 mM, 27.5 mM, and 55 mM were measured and the results are shown inTable 3 (where BFU-3 is Burst-forming unit-erythroid, CFU-G isColony-forming unit-erythroid, CFU-M is colony-forming unit-macrophage,CFU-GM is colony-forming unit-granulocyte, macrophage, and CFU-GEMM iscolony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte)and FIG. 33. Here, cells stored in 27.5 mM (3a) generated far morecolonies (74.5 colonies) than cells sored in DMSO only (31 colonies).These results show a 2.4 fold increase in functionality relative to thecontrol. Individual and total colonies formed (2a) at 5 mM, 8 mM, 12.5mM and 25 mM were measured and the results are shown in Table 4 and FIG.34. Here, cells stored in 25 mM (4a) generated far more colonies (63.0colonies) than cells sored in DMSO only (31 colonies). These resultsshow a 2.0 fold increase in functionality relative to the control.Individual and total colonies formed for (1b) at 11 mM, 18 mM, 27.5 mM,and 55 mM were measured and the results are shown in Table 5 and FIG.35. Here, cells stored in 18 mM (1b) generated far more colonies (56.5colonies) than cells sored in DMSO only (31 colonies). These resultsshow a 1.8 fold increase in functionality relative to the control.Individual and total colonies formed for (4a) at 5 mM, 8 mM, 12.5 mM and25 mM was measured and the results are shown in Table 6 and FIG. 36.Here, cells stored in 12.5 mM (5a) generated far more colonies (47.5colonies) than cells sored in DMSO only (31 colonies). These resultsshow a 1.5 fold increase in functionality relative to the control.

TABLE 3 Number of each type and total colonies formed aftercryopreservation with (3a) at 11, 18, 27.5 and 55 mM post-thaw. Colonieswere scored and counted after 14 days of incubation in Methocult ™media. DMSO Concen- CFU- CFU- Total Number Content IRI tration BFU-ECFU-G CFU-M GM GEMM of Colonies 10% None N/A 15.6 5.1 0.7 6.8 2.8 31.0DMSO (3a)   11 mM 20.5 5.5 0.9 7.4 5.9 40.4   18 mM 18.0 8.8 0.3 11.32.8 36.0 27.5 mM 35.0 6.5 3.5 14.0 15.5 74.5   55 mM 24.5 9.5 1.0 15.06.5 56.5

TABLE 4 Number of each type and total colonies formed aftercryopreservation with (2a) at 5, 8, 12.5 and 25 mM post-thaw. Colonieswere scored and counted after 14 days of incubation in Methocult ™media. DMSO Concen- CFU- CFU- Total Number Content IRI tration BFU-ECFU-G CFU-M GM GEMM of Colonies 10% None N/A 15.6 5.1 0.7 6.8 2.8 31.0DMSO (4a)   5 mM 22.2 4.2 1.3 11.5 6.8 46.0   8 mM 17.0 8.0 0.5 10.5 4.040.0 12.5 mM 44.0 1.5 3.0 8.5 0.0 61.0   25 mM 37.0 11.5 2.0 10.5 2.063.0

TABLE 5 Number of each type and total colonies formed aftercryopreservation with (1b) at 11, 18, 27.5 and 55 mM post-thaw. Colonieswere scored and counted after 14 days of incubation in Methocult ™media. DMSO Concen- CFU- CFU- Total Number Content IRI tration BFU-ECFU-G CFU-M GM GEMM of Colonies 10% None N/A 15.6 5.1 0.7 6.8 2.8 31.0DMSO (1b)   11 mM 25.3 4.3 1.5 9.8 2.4 41.9   18 mM 24.0 9.5 4.5 11.07.5 56.5 27.5 mM 28.5 3.0 2.0 11.0 3.5 48.0   55 mM 29.5 8.5 0.5 10.51.5 50.5

TABLE 6 Number of each type and total colonies formed aftercryopreservation with (4a) at 5, 8, 12.5 and 25 mM post-thaw. Colonieswere scored and counted after 14 days of incubation in Methocult ™media. DMSO Concen- CFU- CFU- Total Number Content IRI tration BFU-ECFU-G CFU-M GM GEMM of Colonies 10% None N/A 15.6 5.1 0.7 6.8 2.8 31.0DMSO (5a)   5 mM 19.2 5.0 1.0 5.2 2.3 32.7   8 mM 18.0 5.0 2.0 4.0 2.031.0 12.5 mM 27.0 8.0 1.5 6.5 4.5 47.5   25 mM 20.5 7.0 2.5 11.0 1.042.0

Human Liver Cell Viability

Human liver cell viability in compound (3a), (4a), (5a), and (1b) wasassessed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) Assay. In the assay, HepG2 cells (human hepatocellularcarcinoma cells) were harvested and incubated overnight at 37° C. in a96-well plate to a concentration of 20,000 cells per well using MinimumEssential Medium (MEM) as media. The following day, the media wasremoved and cells were incubated at 37° C. overnight with 100 μL MEMsupplemented with different concentrations of (3a), (2a), (4a), and(1b). The following day, the plates were centrifuged at 1000 rpm for 5minutes and solutions removed by aspiration. 200 μL of MEM was added toeach well, followed by 50 μL of 2.5 mg/mL MTT (dissolved in HBSS). Theplates were then incubated at 37° C. for 4 hours. After incubation, theplates were centrifuged at 1000 rpm for 5 minutes and solution wasaspirated. 200 μL of a 10% TritonX/0.1 N HCl in isopropanol solution wasadded to each well. The plates were incubated at ambient temperature for3 hours before being shaken and the absorbance at 570 nm of each wellwas read. % viability at 37° C. of (3a) at 11 mM, 22 mM, 44 mM, 55 mMand 110 mM was measured and the results are shown in FIG. 37. This datashows that the concentration of (3a) that yielded the highestfunctionality (see Table 3 and FIG. 33) is minimally cytotoxic relativeto DMSO alone. % viability at 37° C. of (2a) at 11 mM, 22 mM, and 55 mMwas measured and the results are shown in FIG. 38. This data shows thatthe concentration of (2a) that yielded the highest functionality (seeTable 4 and FIG. 34) is minimally cytotoxic relative to DMSO alone. %viability at 37° C. of (1b) at 11 mM, 20 mM, 40 mM, and 55 mM wasmeasured and the results are shown in FIG. 39. This data shows that theconcentration of (1b) that yielded the highest functionality (see Table5 and FIG. 35) is minimally cytotoxic relative to DMSO alone. %viability at 37° C. of (4a) at 0.1 mM, 0.5 mM, 1 mM, 2.5 mM, 5 mM, 10mM, 15 mM, 25 mM, 30 mM, and 40 mM was measured and the results areshown in FIG. 40. This data shows that the concentration of (4a) thatyielded the highest functionality (see Table 6 and FIG. 36) is minimallycytotoxic relative to DMSO alone. Nonetheless, the cytotoxicity of DMSOitself was not determined.

Example 8 Cryopreservation of Three Units of Umbilical Cord BloodSynthesis and Characterization of N-(4-chlorophenyl)-D-gluconamide (5a)

Cryopreservation experiments were performed on three units of UCB, UCBUnit 26, UCB Unit 27, and UCB Unit 28. The UCB units were cryopreservedas described above. Briefly, two cryovials were prepared for eachsample. 50,000 CD34+ cells were aliquoted into cryovials, supernatantremoved and resuspended in 80 uL plasma and 20 uL cryosolution (made a5× concentration, 50% DMSO and various concentration of IRI in 0.9%saline with 5% dextran). Unit 26 was processed and cryopreservedindividually in 11 mM N-(4-methoxyphenyl)-D-gluconamide (3a), 5 mMN-(2-fluorophenyl)-D-gluconamide (2a), 11 mMN-(2,6-difluorobenzyl)-D-gluconamide (1b), and 5 mMN-(4-chlorophenyl)-D-gluconamide (4a). Unit 27 was processed andcryopreserved individually in 11 mM, 18 mM, 27.5 mM and 55 mMN-(4-methoxyphenyl)-D-gluconamide (3a), 11 mM, 18 mM, 27.5 mM and 55 mMN-(2,6-difluorobenzyl)-D-gluconamide (1b), 5, 8, 12.5, and 25 mMN-(2-fluorophenyl)-D-gluconamide (2a), and 5, 8, 12.5, and 25 mMN-(4-chlorophenyl)-D-gluconamide (4a). Unit 28 was processed andcryopreserved individually in 11 mM and 55 mMN-(2-methoxyphenyl)-D-gluconamid (7a), 11 mM and 55 mMN-(3,5-difluorobenzyl)-D-gluconamide (2b), 5 mM and 25 mMN-(4-Fluorophenyl)-D-gluconamide (6a), and 5 mM and 25 mMN-(2-chlorophenyl)-D-gluconamide (5a). Samples were frozen at 1°C./minute to −80° C. before being transferred to a liquid nitrogenstorage dewar. Samples were thawed in a 37° C. water bath before beingdiluted with 0.9 mL of IMDM (supplemented with 10% FBS and 1% pen/strep)for a CD34+ concentration of 50,000 cells/mL. These samples were quicklytaken to the hospital for CFU analysis.

CFU assays were performed as described above. Briefly, cryovials wereobtained that contain 1 mL 50,000 CD34+ cells/mL. The sample is mixed bypipetting and 80 uL is transferred to a 15 mL falcon tube, diluted with5 mL IMDM (sup. 10% FBS), centrifuged for 6 minutes at 1100 rpm andsupernatant removed by aspiration. The pellet is resuspended in 1 mL ofIMDM (sup. 2% FBS) and pipetted to mix. 150 uL of the washed cells isadded to 3 mL of Methocult® media and vortexed to mix. Upon settling, 1mL is added to 2 plates for culturing (therefore, one cryovial is splitinto 2 plates). Overall, 190 CD34+ cells are seeded. After 2 weeks ofculture, the colonies which have formed are identified by type andquantified.

The results for units 26, 27, and 28 were combined and averaged. Theresults of a post-thaw viability analysis are provided in Table 7, whichprovides the percent viability resulting from controls and in Table 8,which provides the percent viability resulting from IRIs (3a), (1b),(2a), and (4a). This data shows that the CD34+ cells stored in the IRIshad a percent viability that was similar to the percent viability ofCD34+ cells stored in the control solutions.

TABLE 7 Percent viability results for CD34+ cells cryopreserved incontrol solutions Pre-freeze Post-Thaw Tot. No. Cryosolution UCB UnitViab. Viab. Colonies 10% DMSO (⅕ 26 89% Not measured 10 dilution with 10plasma)) 23 27 27 97% 88% 56 35 28 94% 87% 36 36 10% DMSO (no 83% 47plasma) 34 AVG: 28

TABLE 8 Percent viability results for CD34+ cells cryopreserved in IRIs.Post-Thaw Tot. No. Cryosolution UCB Unit Pre-Freeze Viab. Viab. Colonies11 mM 26 89% Not measured 33 56 26 25 27 97% 78% 33 37 11 mM 26 89% Notmeasured 58 63 27 39 27 97% 57% 31 30  5 mM (2a) 26 89% Not measured 4866 42 48 27 97% 88% 38 34  5 mM (4a) 26 89% Not measured 45 42 14 10 2797% 88% 47 38

The combined results of the CFU assays are shown in the graph of FIG.41. In the graph, a horizontal line has been placed at the level of thecontrol. Therefore, bars that extend beyond the horizontal linerepresent the concentration of a particular IRI that performs betterthan the control. As shown in the graph, the IRIs (3a), (2a), (4a), and(1b) performed particularly well. In contrast, compounds (5a), (6a),(7a), and (2b) did not perform particularly well. These data demonstratethat that IRIs, such as compounds (3a), (2a), (4a), and (1b), are anecessary supplement for improving functionality of cryopreservedumbilical cord blood or hematopoietic stem cells. Additionally, as shownabove, these IRIs have a low cytotoxicity at the concentrations thatprovide the best results.

1. A composition for cryopreserving umbilical cord blood, thecomposition comprising: at least one ice recrystallization inhibitor(IRI) compound, wherein the IRI is a benzyl-aldonamide.
 2. Thecomposition of claim 1, wherein the benzyl-aldonamide corresponds instructure to Formula B′

wherein R and R₂ are independently a hydrogen; C₁-C₄-alkoxy; or haloselected from the group consisting of Br, Cl, and F, and n is a wholenumber from 1 to
 20. 3. The composition of claim 2, wherein thebenzyl-aldonamide corresponding in structure to Formula B′ is

N-(2,6-difluorobenzyl)-D-gluconamide (1b)
 4. The composition of claim 3,wherein the N-(2,6-difluorobenzyl)-D-gluconamide is generated by:combining D-gluconic acid-d-lactone with 2,6-difluoroaniline in asolvent to generate a mixture; stirring the mixture under reflux forfrom about 12 hours to about 48 hours; evaporating the solvent togenerate a residue; and recrystallizing the residue to yield theN-(2,6-difluorobenzyl)-D-gluconamide.
 5. The composition of claim 4,wherein the combining D-gluconic acid-d-lactone with 2,6-difluoroanilinecomprises combining D-gluconic acid-d-lactone with 2,6-difluoroanilineat a D-gluconic acid-d-lactone:2,6-difluooaniline ratio of from about5:1 to about 1:5.
 6. The composition of claim 5, wherein the D-gluconicacid-d-lactone:2,6-difluooaniline ratio is about 1:1.
 7. The compositionof claim 4, wherein the combining D-gluconic acid-d-lactone with2,6-difluoroaniline in a solvent comprises combining D-gluconicacid-d-lactone with 2,6-difluoroaniline in methanol.
 8. The compositionaccording to claim 1, further comprising: at least one cryopreservationagent.
 9. The composition according to claim 8, wherein the at least onecryopreservation agent is selected from the group consisting of DMSO,glycerol, polyvinyl alcohol, other biopolymers, or a combinationthereof.
 10. A method for cryopreserving umbilical cord blood, themethod comprising: fractionating whole umbilical cord blood to generatea fraction comprising hematopoietic stem cells; mixing the hematopoieticstem cells in a solution comprising at least one ice recrystallizationinhibitor (IRI) compound to form an IRI suspension, wherein the IRI isan unsubstituted aryl-aldonamide, a mono-substituted aryl-aldonamide, ora di-substituted aryl-aldonamide; and freezing the IRI suspension. 11.The method according to claim 10, wherein the suspending the fraction ina solution comprising at least one IRI compound to form a suspensioncomprises suspending the fraction in a solution comprising at least oneIRI compound corresponding in structure to: Formula B:

wherein R is hydrogen; C₁-C₄-alkoxy; or halo selected from the groupconsisting of Br, Cl, and F; Formula B′:

wherein R and R₂ are independently a hydrogen; C₁-C₄-alkoxy; or haloselected from the group consisting of Br, Cl, and F; and n is a wholenumber from 1 to 20; or a combination thereof.
 12. The method accordingto claim 11, wherein the at least one IRI compound corresponding instructure to Formula B is

and the at least one IRI compound corresponding in structure to FormulaB′ is


13. The method according to claim 10, wherein prior to the mixing thehematopoietic stem cells in a solution comprising at least one IRIcompound, the method further comprises: transferring the fraction into acryovial; centrifuging the cryovial to generate a pellet comprising thehematopoietic stem cells and a supernatant; removing the supernatant;and adding plasma to the hematopoietic stem cells to form a suspensioncomprising the hematopoietic stem cells.
 14. The method according toclaim 10, wherein the freezing the IRI suspension comprises: cooling theIRI suspension at a cooling rate of about −1° C. per minute in a −80° C.freezer for 24 hours to freeze the IRI suspension; and transferring thefrozen IRI suspension to a liquid nitrogen dewar for storage.
 15. Themethod according to claim 10, wherein mixing the hematopoietic stemcells in a solution comprising at least one IRI compound to form an IRIsuspension comprises adding N-(2,6-difluorobenzyl)-D-gluconamide (1b) tothe hematopoietic stems cells to a finalN-(2,6-difluorobenzyl)-D-gluconamide (1b) concentration of from about 10mM to about 55 mM, and mixing to form the IRI suspension.
 16. The methodaccording to claim 10, wherein mixing the hematopoietic stem cells in asolution comprising at least one IRI compound to form an IRI suspensioncomprises adding N-(4-methoxyphenyl)-D-gluconamide (3a) to thehematopoietic stems cells to a final N-(4-methoxyphenyl)-D-gluconamide(3a) concentration of from about 10 mM to about 55 mM, and mixing toform the IRI suspension.
 17. The method according to claim 10, whereinmixing the hematopoietic stem cells in a solution comprising at leastone IRI compound to form an IRI suspension comprises addingN-(2-fluorophenyl)-D-gluconamide (2a) to the hematopoietic stems cellsto a final N-(2-fluorophenyl)-D-gluconamide (2a) concentration of fromabout 5 mM to about 25 mM, and mixing to form the IRI suspension. 18.The method according to claim 10, wherein mixing the hematopoietic stemcells in a solution comprising at least one IRI compound to form an IRIsuspension comprises adding N-(4-chlorophenyl)-D-gluconamide (4a) to thehematopoietic stems cells to a final N-(4-chlorophenyl)-D-gluconamide(4a) concentration of from about 5 mM to about 25 mM, and mixing to formthe IRI suspension.
 19. A method for cryopreserving hematopoietic stemcells, the method comprising: adding an ice recrystallization inhibitor(IRI) compound to a suspension of hematopoietic stem cells to form anIRI suspension; and freezing the IRI suspension, wherein the IRIcompound corresponds to Formula B′:

wherein R and R₂ are independently a hydrogen; C₁-C₄-alkoxy; or haloselected from the group consisting of Br, Cl, and F, and n is a wholenumber from 1 to
 20. 20. The method according to claim 19, wherein theIRI compound is N-(2,6-difluorobenzyl)-D-gluconamide corresponding tostructure (1b):


21. The method according to claim 20, wherein theN-(2,6-difluorobenzyl)-D-gluconamide is added to a final concentrationof from about 10 mM to about 55 mM.
 22. A composition comprisingN-(2,6-difluorobenzyl)-D-gluconamide having structure (1 b):